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Differences in the expression of opioid peptides in Dahl salt-resistant and salt-sensitive rats Hao, Jing-Ming 1993

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DIFFERENCES IN THE EXPRESSION OF OPIOID PEPTIDESIN DAHL SALT-RESISTANT AND SALT-SENSITIVE RATSbyJING-MING HAOB.Med., Shandong Medical University, China, 1982M.Med., Shandong Medical University, China, 1987A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENT FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Medicine)We accept thi thesis as conformingto th equired standardTHE UNIVE ITY OF BRITISH COLUMBIAMay 1993© Jing-ming Hao, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of M ech c241 e The University of British ColumbiaVancouver, CanadaDateDE-6 (2/88)iiABSTRACTThe Dahl strain of genetically salt-resistant (R) and salt-sensitive (S) rats affords anopportunity to explore mechanisms for the development of hypertension and theconsequences, as well as sensitivity or resistance, of salt-induced hypertension. Becauseof the evidence that opioid peptides and their receptors can be involved in cardiovascularregulation, the objective of this study was to test the hypothesis that /3-endorphin andenkephalins are involved in the development of hypertension through the determinationof their precursors preproopiomelanocortin (POMC) and preproenkephalin (ppENK)messenger RNAs in this animal model. Three week old inbred Dahl R and S rats weremaintained on high salt diet (8% NaCI) or low salt diet (0.4%) for six weeks. POMCmRNA and ppenk mRNA were examined from tissues of Dahl R and S rats as determinedby Northern blot analysis using p-actin as an internal standard. POMC mRNA wasabundant in the pituitary tissues and was not detectable in other tissues. ppenk mRNAwas abundantly present in brain, testis and heart. There was more POMC mRNA in thepituitary tissue of R rats on high salt diet compared with the pituitary of S rats on high saltdiet. Differences in POMC mRNA in the pituitary were not observed between R and S onlow salt diet. There were no differences of ppENK mRNA in the brain between R and Srats on high salt diet. Increased ppENK mRNA was found in the right and left ventriclesof the heart of S rats on low salt diet. The increase in ppENK mRNA in the cardiacventricles of S rats was exaggerated when they were on high salt diet. To define the roleof increased ppENK mRNA in the S rats in the regulation of cardiovascular function, theeffects of intravenous administration of proenkephalin-derived bioactive peptides Leu5-enkephalin (LE), Met5-enkephalin (ME), Met5-enkephalin-Arg6-Gly7-Leu5 (MEAGL), andMet5-enkephalin-Ard-Phe7 (MEAP) in Sprague-Dawley rats, and effects of these peptideson isolated intact hearts of Sprague-Dawley rats, were examined. At intravenous dosesof 3.6 and 36 nmole, none of the opioid peptides had effects on arterial pressure or heartrate. Intravenous administration of LE, ME, MEAGL or MEAP at a dose of 360 nmole peranimal briefly deceased the heart rate. The MEAP, but not LE, ME, or MEAGL, induceda more prolonged increase in arterial pressure at the intravenous dose of 360 nmole. LE,ME, MEAGL or MEAP, at concentration 10-6 M in the perfusion solution, had no directeffect on the developed pressure in the left ventricle of the isolated heart. These datashow increased preproenkephalin mRNA in the heart and decreased POMC mRNA in thepituitary of Dahl S compared to Dahl R rats. From these results we speculate that: (1)inefficient pituitary production of POMC and consequently J3-endorphin, which is knownto decrease arterial pressure, may contribute to the development of hypertension in theS rats on high salt diet; (2) increased release from the heart of the opioid peptide MEAP,and possibly other enkephalins, may be related to the high blood pressure of S rats onlow salt diet; (3) exaggerated release of MEAP and/or other enkephalins may exacerbatethe process of hypertension in S rats on high salt diet.Table of ContentsPageABSTRACT^Table of Contents  ivList of TrA bl ec^  viiList of T2i9i;tve> ViAcknowledgement^INTRODUCTION  1I.^Endogenous opioid peptides and their receptors^  11.1. Endogenous opioid peptides and their precursors  11.2. Receptors for opioid peptides^  3Regulation of blood pressure and sodium in hypertension^ 411.1. Mechanisms for control of blood pressure^  411.2. Sodium and hypertension^  511.3. Dahl salt-sensitive rats - an animal model for hypertension^ 7III. Opioids and cardiovascular system^  7111.1. Opioids in the regulation of blood pressure^  7111.2. Endogenous opioid peptides and hypertension  8111.3. Opioid peptides and receptors in the heart^  9IV. Rationale and objectives^  11ivVMATERIAL AND METHODS^  13I. Effect of intravenous administration of proenkephalin-derivedpeptides LE, ME, MEAGL and MEAP^  131.1. Animals^  131.2. Method  13II. Effects of proenkephalin-derived peptides on isolated intact heart^ 1411.1. Method^  1411.2. protocol  15III. Northern blot analysis of POMC and ppENK mRNAin various tissues of Dahl R and S rats^  15111.1. Animals^  15111.2. Isolation of total RNA^  15111.3. Complementary DNA probes^  16111.4. RNA electrophoresis and Northern blot analysis^ 17IV. Materials^  18RESULTS^  19I. Effects of intravenously administered enkephalinsin the unanesthetized Sprague-Dawley rats^  19II. Effects of enkephalins on isolated hearts of Sprague-Dawley rats^ 25III.^Northern blot analysis: decreased expression of POMCand increased expression of ppENK in S rats^  25111.1. Ventricular hypertrophy in S rats^  25vi111.2. Increased expression of ppENK mRNAin the cardiac ventricles of S rats^  28111.3. Increased expression of POMC messenger RNAin R rats on high salt diet^  28DISCUSSION^  42I. Effects of intravenous administration of enkephalins^ 43II. The effect of enkephalins on the isolated intact heart  44III. Transcription of POMC and ppENK genes in different tissues^ 46111.1. Messenger RNA for ppENK^  46111.2. Messenger RNA for POMC  47IV. Differences in the expression of ppENK and POMC in R and S rats ^ 481V.1. Increased ppENK mRNA in the cardiac ventricles of S rats^ 48IV.2. Increased POMC mRNA in the pituitary of R ratson high salt diet: responsible for salt resistant?^ 52V.^Summary^  54REFERENCES  57list of TableTable^ Page1. Heart and body weight of R and S rats on high and low salt diet^ 27List of FiguresFigure^ Page1. Change in heart rate after intravenous administration ofproenkephalin-derived peptides of different doses^  202. Change in mean arterial pressure after intravenous administration ofproenkephalin-derived peptides of different doses^  213. Time course of heart rate after intravenous administration ofproenkephalin-derived peptides^  224. Change in heart rate with respect to time after intravenous administrationof proenkephalin-derived peptides^  235. Change in mean arterial pressure with respect to time after intravenousadministration of proenkephalin-derived peptides^  246. Effects of proenkephalin-derived peptides on developed pressure ofthe left ventricle in isolated heart^  267. Expression of ppENK in different tissues of rats^  298. ppENK mRNA in different brain regions of R and S rats  309. ppENK mRNA in cardiac tissues of R and S rats on high salt diet^ 3110. ppENK mRNA in cardiac tissues of R and S rats on low salt diet  3211. fi -Actin mRNA in cardiac tissues as standard controls^  3312. Comparison of ppENK mRNA between R and S rats on high and low salt diet^ 3413. Ratio of heart/body of R and S rats used for cardiac ppENK mRNA analysis^ 3514. Expression of POMC mRNA in different tissues of rats^  3715. POMC, ppENK, p -actin mRNA in the pituitary of R and S rats on high salt diet^ 3816. Densitometric comparison of POMC, ppENK, /3-actin mRNA in the pituitaryof R and S rats on high salt diet^  3917. POMC mRNA in the pituitary of R and S rats on high and low salt diet^ 4018. Densitometric comparison of POMC mRNA in the pituitary of R and S ratson high and low salt diet^  4119. Schematic summary  56xAcknowledgementI would like to thank Dr. Simon W. Rabkin for his guidance and support throughoutthis work. I am grateful to Carol Smythe, Frederick L. Carranza, and Paul S. Sunga fortheir technical assistance.1INTRODUCTIONEndogenous opioids refer to the group of peptides that exist in the body and havemorphine-like biological activities. In less than two decades since the first discovery ofmet- and leu-enkephalin, opioid peptides have been found to be closely involved in theregulation of pain perception, behaviour, reproduction, gastrointestinal function andimmune system, acting as neurotransmitters, neuroendocrinal hormones and localchemical mediators (Burks 1989; Carr 1991; Hawkes 1992; Laatikainen 1991). There hasalso been increasing evidence that opioid peptides participate in the modulation of thecardiovascular system, and the change in the opioid system may be related to thepathogenesis of cardiovascular conditions such as hypertension (Feuerstein and Siren1987). The exact roles played by different opioid peptides and opioid receptors invarious types of hypertension have not been completely defined.I.^Endogenous opioid peptides and their receptors1.1. Endogenous opioid peptides and their precursorsIn a search for the endogenous morphine-like substance, two related pentapeptideswith potent opiate agonist activity were first identified in 1975 (Hughes et al. 1975). Thetwo peptides differed only in the carboxyl terminal amino acid and were namedmethionine-enkephalin and leucine-enkephalin (with methionine or leucine as amino acidin the carboxyl end fifth position). The name enkephalin was derived from en kephalos,2meaning 'in the head' because they were first discovered in the brain. The generic nameendorphin was also used with endo signifying endogenous and orphin, a common suffixin the names of opioids. This is why today endogenous peptides are still sometimescalled endorphins. Since then numerous other peptides have been found to possess theopioid activity. Most of these peptides are C-terminal extensions of either Met-enkephalinor Leu-enkephalin. For example, p-endorphin is a C-terminal extension of Met-enkephalin,while p-neo-endorphin and dynorphin are C-terminal extensions of Leu-enkephalin.Almost all the opioid peptides discovered so far are derived from three precursorpolyproteins - proopiomelanocortin (POMC), proenkephalin, and prodynorphin (Kitchen1985). The main opioid products of POMC are 0-endorphin and a-endorphin.Proenkephalin has four well-known bioactive peptide products, Met5-enkephalin(methionine-enkephalin or ME), Leu5-enkephalin (leucine-enkephalin or LE), Met5-enkephalin-Arg6-Gly7-Leu5 (MEAGL), and Met5-enkephalin-Arg6-Phe7 (MEAP). The mainprodynorphin-derived products are dynorphin A, dynorphin B, a-neo-endorphin and p-neo-endorphin. By a convention, the endogenous opioid peptides derived from POMC,proenkephalin, and prodynorphin are called endorphins, enkephalins and dynorphinsrespectively (Feuerstein and Siren 1987). The opioid peptide precursors showremarkable similarities at the structural level (Cochet et al. 1982; Comb et al. 1982;Horikawa et al. 1983; Noda et al. 1982). They are almost all exactly the same length. Thebiologically active peptides are confined to the C-terminal half of each protein while all ofthe cysteine residues are concentrated in the N-terminal region of the proteins.The similarity in structure of the genes for three precursor polyproteins is equally3remarkable. They all contain large 3' exons that code for 80% of the translated regionof the mRNA. A large intron then separates this exon from a smaller exon that codes forthe signal sequence and about 45 other amino acids in the N-terminal of eachpolyprotein. The second intron separates the signal sequence exon from an exon orexons that code for 5' untranslated region (Cochet et al. 1982; Comb et al. 1982;Horikawa et al. 1983; Noda et al. 1982). The structural similarities among these threegenes suggest that they arose via similar evolutionary mechanisms.1.2. Receptors for endogenous opioid peptidesAt least five types of opioid receptors have been defined. They are p„ 6, K, C, ande receptors. A, 6, K and a receptors have been further subclassified into pi and I/2; 61and 62; Ki, ic,2 and K3; Ci and cr2 subtypes (Pasternack 1993; Quirion et al. 1992). Amongthe most investigated endogenous opioid peptides, 0-endorphin is predominantly a Aagonist and an E agonist, enkephalins are mainly 6 and A agonists with greater affinity for6 receptors than for A receptors, dynorphins are preferentially K agonists (Feuerstein andSiren 1987a). Opioid receptors in vascular tissues are most likely of 6, K, and A types(Hughes 1980). But E opioid receptors are also reported in the vascular tissues and /3-endorphin is the natural agonist for E receptor (Bucher et al. 1987; Schulz et al. 1981).Opioid receptors in cardiac sarcolemma are mainly 6 and K types, but the presence ofa A opioid receptor in the heart was also observed (Krumins 1987; Krumins et al. 1985;Ventura et al. 1989). The role of opioids in blood pressure regulation and salt-dependenthypertension and its consequences on the heart will be discussed.4II.^Regulation of blood pressure and sodium in hypertension11.1. Mechanisms for control of blood pressureThe arterial pressure required to move blood through the circulatory bed is providedby pumping action of the heart (cardiac output) and the tone of the arteries (peripheralresistance). The former is influenced by the volume of blood returning to the heart(preload) and cardiac contractility. Each of these primary determinants is, in turn,influenced by the interaction of an exceedingly complex series of factors. The main areasinvolved in the regulation include neural, humeral, and renal factors. The vasomotorcentre, located in the reticular substance of the medulla and the lower third of the pons,regulates the vascular tone and heart contractility through sympathetic nerve fibres, andinfluences the heart rate by sympathetic and parasympathetic nerve fibres. Sympatheticstimulation increases the tone of arteries, heart rate and contractility--and consequentlyarterial blood pressure--while parasympathetic drive decreases the heart rate and, to alesser extent, blood pressure. The vasomotor centre is controlled by higher nervouscentres in hypothalamus, hippocampus, amygdala, cingulate gyrus, and other brain areasThis centre is also modulated by the afferent message from baroreceptors andchemoreceptors in the carotid and aortic arteries, cardiac chambers and pulmonaryarteries. Numerous humeral factors can regulate blood pressure by changing vasculartone, cardiac contractility, and preload (mostly through renal sodium excretion). Humeralfactors are also responsible for vascular hypertrophy, a final pathway in the pathogenesisof hypertension. Among the organ systems, the kidneys play a dominant role in the long-term regulation of arterial blood pressure through their control over sodium excretion and5body fluid volume. An important mechanism for the kidney to influence blood pressureis the phenomenon of pressure-natriuresis - that is, in normal circumstances, when theblood pressure rises, renal excretion of sodium and water increases, shrinking fluidvolume and returning the pressure to normal. According to the pressure-natriuresisrelationship, if fluid volume and cardiac output are high in the presence of an elevatedblood pressure, something must be at fault in the renal-body fluid pressure controlmechanism and the resetting of pressure-natriuresis is held to be responsible for thedevelopment of hypertension (Guyton 1987).11.2. Sodium and hypertensionThe presence of excess sodium - within the circulation or within the cell or both - iswidely held to be a necessary, though not an exclusive, basis for the development ofprimary hypertension. The resetting of the pressure-natriuresis curve that prevents ashrinkage of fluid volume from self-correcting. A rise in blood pressure may be lookedupon as essential for the maintenance of hypertension, regardless of whatever initiatesthe rise in pressure. However, the renal retention of sodium may be the fundamentaldefect and may not play just a supporting role. Based on their work on salt-sensitive rats,Dahl and co-workers proposed that hypertension might evolve as follows: high sodiumintake, together with genetic defect in renal sodium excretion, increases body fluid andconsequently blood volume. This increases cardiac output, which, through local tissueautoregulation of blood flow or the increase in an unidentified 'sodium excretinghormone', induces increased peripheral resistance, and results in hypertension (Dahl et6al. 1969). The existence of a natriuretic hormone, which is proposed as a sodium pumpinhibitor (de Wardener and MacGregor 1982, 1983), has been under investigation.Evidence for its existence was initially indirect, namely, the exaggerated natriuresis whenhypertensive individuals were given an acute sodium load (Baldwin et al. 1958). Theinhibition of sodium pump or other abnormalities in the sodium transportation thatincrease intracellular sodium concentration produce increased intracellular calciumthrough enhanced Na+-Ca2+ exchange (Blaustein 1988; Blaustein et al. 1986). Increasedfree calcium in the cytosol is thought to be responsible for increased vascular tone andcardiac contractility. There exists evidence for enhanced sodium influx and reduced effluxin the cells from hypertensive patients (Edmundsun et al. 1975; Ng et al. 1988).One other mechanism for higher intracellular sodium in hypertensives is theincreased Na±/H+ antiport (Ng 1989). One of the major functions of Nal-/H+ exchangemay be the control of intracellular pH. If Na+/H+ exchange and sodium influx areincreased, intracellular pH would be expected to be higher, i.e., more alkaline. Theregulation of cell pH in turn is intimately involved with the control of cell growth andproliferation (Pouyssegur 1985). Growth factors such as angiotensin ll can activate theantiporter in vascular smooth muscle cells so that the cytoplasm becomes more alkalineand cell growth is stimulated (Vallega et al. 1988). Not only would such a higher pHstimulate cell growth, but it would also raise cytosolic free calcium by releasing calciumfrom intracellular stores and enhance the affinity of myosin for calcium, increasing thesensitivity of smooth muscle to vasoconstrictors (Siskind et al. 1989; Danthuluri and Deth1989).711.3. Dahl salt-sensitive rat - an animal model for hypertensionTwo genetically different strains of rats developed by Dahl et al. have provided amodel of salt dependent hypertension (Dahl et al. 1968; Rapp 1984). The Dahl salt-sensitive (S) strain rapidly develops severe hypertension on a high salt diet while itdevelops hypertension at slower rate on low (0.3% NaCI) or normal (1%) salt diet, but theDahl salt-resistant (R) strain remains normotensive whether on low or high salt diet (Dahlet al. 1968; Rapp 1984). The mechanism of this salt-dependent hypertension has notbeen completely elucidated. The roles of unknown humeral factors and renalabnormalities in the pathogenesis were defined in the early observations (Dahl et al. 1969;Dahl and Heine 1975). Recent genetic studies have found that renin and atrial natriureticpeptide (ANP) receptor gene polymorphisms of Dahl rats cosegregates with bloodpressure (Deng and Rapp 1992; Rapp et al. 1989). In addition, afferent baroreceptors,as well as central neural and peripheral adrenergic mechanisms, have been implicatedin the pathogenesis (Mark 1991), but the mediators have not been identified.III. Opioids and cardiovascular system111.1. Opioids in the regulation of blood pressureThe effects of opiates such as morphine on the heart and blood pressure have beenknown for a long time (Jackson 1914). Considerable evidence has also accumulatedimplicating roles of endogenous opioids in blood pressure regulation. Opioid peptidesand their receptors are present in brain areas important for cardiovascular control, inheart, in autonomic ganglia, and in adrenal gland (Goodman et al. 1980; Hahnbauer et8al. 1982; Khachaturian et al. 1985; Krumins 1987; Schultzberg et al. 1979). Microinjectionof various opioids into certain areas in the brain can profoundly alter blood pressure(Feuerstein 1985; Rabkin 1989). Opioid peptides also have potent effects on arterialblood pressure and heart rate when administered systemically (Douglas and Kitchen1992; Lemaire et al. 1978; Moore and Dowling 1980). Moreover, endogenous opioidshave been shown to have direct action on the heart and vascular tissue in vitro, thoughthe responses were not the same in different tissue preparations (Eiden and Ruth 1982;Gillespie et al. 1984; Ruth et al. 1984, 1984a; Sun and Zang 1985). More interestingly,enkephalins and analogues modulate baroreceptor reflex in animals (Gordon 1986;Matsumura et al. 1992; Petty and Reid 1982; Schaz et al. 1980; Yukimura et al. 1981).III.2.Endogenous opioids and hypertensionHypertension is associated with significant changes in the opioid system. Evidencelinking opioid system to hypertensive state first appeared as a result of investigations ofpain sensitivity. Neuroanatomical as well as neurophysiological evidence has shown thatboth central cardiovascular and pain regulating systems are closely related and, in fact,these systems share common central nervous system loci that include the nucleus of thesolitary tract and nuclei of the vagus nerve (Akil and Liebeskind 1975; Chalmers 1975).Opioid peptides and receptors have been known to be present in these brain areas(Armstrong et al. 1981; Martucci and Hahn 1979). Besides, hypalgesia has beendemonstrated in various models of experimental and genetic hypertension such asrenovascular hypertensive rats, DOCA-salt hypertensive rats, Dahl salt-sensitive rats and9the spontaneous hypertensive rats (SHR) (de Jong et al. 1983; Friedman et al. 1984;Zamir and Segal 1979; Zamir et al. 1980). It is interesting to note that decreased painsensitivity has also been observed in humans with essential hypertension (Ghione et al.1988; Zamir and Shuber 1980). Because the endogenous opioid system is closelyinvolved in pain perception, it is reasonable to believe that changes in this system arealso involved in the evolution of hypertension. Direct evidence for the changes in opioidsystem in hypertensive animals and humans is also available. The levels of dynorphinsand leu-enkephalin in the pituitary gland and several brain areas are substantially differentin spontaneously hypertensive rats (SHR) compared with normotensive animals(Feuerstein et al. 1983). Preproenkephalin messenger RNA levels may be increased inhypothalamus, midbrain, cerebellum, and thoracic cord, and decreased in pons-medullaof SHR relative to the normal controls (Hoegler et al. 1989). Alterations in the brain opioidreceptors in both experimental and genetic hypertension have also been observed (Hahn1985; Zamir et al. 1980a, 1981). Low plasma levels of /3-endorphin were observed inseveral patient groups with essential hypertension and decreased blood pressure afterclonidine administration in these patients was accompanied by normalization of plasmap-endorphin concentration (Kraft et al. 1987, 1988).III.3.0pioid peptides and receptors in the heartProenkephalin and prodynorphin derived peptides were found to be present in theheart (Dumont et al. 1991; Lang et al. 1982; Weihe et al. 1985). Preproenkephalintranscription is developmentally regulated in the cardiac ventricle (Dumont et al. 1991;10Springhorn and Claycomb 1989) and ventricular tissue has the richest preproenkephalinmRNA content among tissues from adult rats (Howells et al. 1986). There exists adiscrepancy between the cardiac levels of preproenkephalin mRNA and the expressedpeptides (Dumont et al. 1991; Howells et al. 1986). One explanation is that ventricularcardiomyocytes are unable to store biosynthetic peptide products. (Bloch et al. 1986).Another interesting phenomenon is that peptides exist mostly under precursor forms inthe ventricular cardiomyocytes (Dumont et al. 1991; Howells et al. 1986). But thepresence of opioid receptors and small amounts of bioactive opioid peptides alsosuggests the concept of local function (Dumont and Lemaire 1988; et al. 1991; Krumins1987; Mantelli et al. 1987; Weihe et al. 1985). The opiate receptors in the heart are mainlyof 6, and K types (Krumins 1987; Krumins et al. 1985). Both s and K agonists wereshown to increase inositol 1,4,5-triphosphate in the cardiomyocyte and to cause Ca2+release and depletion from intracellular store sites, which results in reduced contractility(Ventura et al. 1992). K-Opioid receptor stimulation was also found to increase cytosolicpH and myofilament responsiveness to Ca2+ in cardiac myocyte most likely via proteinkinase C activation of amiloride-sensitive Na+/H+ exchanger (Ventura et al. 1991). Thetranscription of ppENK mRNA in the ventricular tissue has been found to be increasedin some pathological conditions such as hypertension with cardiac hypertrophy,cardiomyopathy, and myocardial infarction (Dumont et al. 1991; Ouellette and Brakier-Gingral 1988; Paradis et al. 1992). The down-regulation low-affinity of 6-receptor was alsonoted in the hearts of spontaneously hypertensive rats (Dumont and Lemaire 1988).11IV. Rationale and ObjectivesThe hypothesis to be examined was that endogenous opioids are involved in saltdependent hypertension and blood pressure regulation. The opioid genes aretranscribed in the cell nucleus into messenger RNAs, which are transported ontoribosomes of endoplasmic reticulum and translated into polyprotein precursors, which areprocessed subsequently in the endoplasmic reticulum, Golgi apparatus, and secretoryvesicles into bioactive peptides, and are exocytosized (constitutive secretion) or storedin the secretory vesicles and secreted at releasing signals (regulatory secretion)(Douglass et al. 1984; Smith and Funder 1988). The released opioid peptides will actlocally as neurotransmitters or other local chemical mediators, or be transported throughthe circulatory system to the target tissues. Transcription is the usual rate-limiting stepfor the peptide biosynthesis. The measurement of messenger RNA levels can providea reflection of relative transcription rate. Messenger RNAs coding for three opioidpeptide precursors, pre-proomiomelanocortin (POMC), pre-proenkephalin (ppENK) andpre-prodynorphin (ppDYN), have been found in various tissues (Douglass et al. 1984;Howells et al. 1986; Jingami et al. 1984). In this study, we used complimentary DNAs forPOMC and ppENK messenger RNAs as probes to tissue RNA from Dahl salt-sensitiveand salt-resistant rats in order to test the hypothesis that change in the production andsecretion of opioid peptides may be closely involved in the pathogenesis of saltdependent hypertension. To relate potential changes in opioid polypeptide synthesis tofunctional effects, the action of opioids on the isolated heart and blood pressure in theintact animal were examined.The specific aims were:(1) to determine effects of four proenkephalin products, Leu-enkephalin (LE),Met-enkephalin (ME), MEAGL and MEAP, on blood pressure whenadministered intravenously;(2) to determine effects of the four proenkephalin-derived peptides on cardiaccontractility;(3) to examine the expression of enkephalins in the hearts of rats with saltdependent hypertension and cardiac hypertrophy;(4) to examine POMC and ppENK messenger RNA expression in brain andother tissues from Dahl salt sensitive and salt-resistant rats.1213MATERIALS AND METHODSI.^Effect of intravenous administration of proenkephalin-derived peptides LE, ME,MEAGL and MEAP1.1. AnimalsSprague-Dawley male rats at ten weeks old, 280-300 g, were obtained from AnimalCare Centre, University of British Columbia (originally from Charles River, St. Constant,Quebec, Canada).1.2. MethodCatheters, PE-50 polyethylene tubing (Clay Adams of Becton Dickinson andCompany, Parsippany, New Jersey, USA), were implanted under halothane anaesthesiainto the femoral artery and femoral vein and tunnelled under skin to exit through the skinon the back of the neck. The animals were then allowed to recover from anaesthesia for1 hour before intravenous administration of enkephalins. The animals were permitted tomove freely and arterial blood pressure was monitored through the femoral arterycatheter connected to a Gould Statham Transducer P23ID and Gould Recorder 2400polygraph (Gould Inc., Cleveland, Ohio, U.S.A.). The peptides were initially dissolved insterile water, then diluted in 0.9% saline and administered through the catheter in thefemoral vein. Each animal received as bolus 0.5 ml of 0 (control injection of the diluent0.9% saline), 3.6, 36, and 360 nmoles in a sequential manner. The doses were selectedfrom the work of Schaz et al (Schaz et al. 1980) who reported that these were effective14doses for the unanaesthetized Wistar-Kyoto rat. After each bolus administration, thecatheter was flushed with 1 ml 0.9% saline. Arterial blood pressure was recorded at thepaper speed of 5 mm/second at the time of peptide administration. The doses wereadministered at 30 minute intervals.II.^Effect of proenkephalin-derived peptides on isolated heart11.1. MethodSprague-Dawley male rats, ten weeks of age, were anaesthetized with halothane andsacrificed by cervical dislocation. The heart was quickly excised and placed inoxygenated Krebs-Henseleit solution. The aorta was then immediately cannulated andthe heart perfused by Langendorff technique with oxygenated Krebs-Henseleit solutionat a speed of 12 ml/min. The perfusing solution had the following composition (inmmol/L): NaCI, 119.9; NaHCO3, 25.0; KCI, 6.0; KH2PO4, 1.2; CaCl2, 1.6; MgSO4, 1.2; anddextrose, 10.0. The perfusate was previously equilibrated and constantly aerated with95% 02 and 5% CO2 and kept at 37°C. The right ventricle was stimulated with squarewaves of 1 V for 1 ms every 300 ms (Pulsar 61 stimulator, Frederick Haer & Co.,Brunswick, Maine, USA). The left atrium was incised to permit the insertion of a catheter,with a small latex tip, through the atrial chamber into the left ventricle. The balloon wasinflated to a resting tension of 20 mm Hg. Left ventricular pressure was measured usinga Gould Statham pressure transducer P23ID and recorded on a Gould Recorder 2400polygraph (Gould Inc. Cleveland, Ohio, USA). The preparation was allowed to equilibratefor 20 minutes prior to commencement of the experimental protocol.1511.2. ProtocolThe heart was sequentially perfused with the above solution containing Leu-enkephalin, Met-enkephalin, MEAP and MEAGL at a concentration of 10-6 M. Each opioidpeptide perfusion lasted for 3 minutes and 15 minutes peptide-free solution was perfusedbetween different peptides and different concentrations. The order of sequence fordifferent enkephalin administration was altered for each isolated heart.III. Northern blot analysis of POMC and ppENK mRNA in various tissues of Dahl R andS rats111.1. AnimalsMale inbred Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats were obtainedfrom Harlan Sprague-Dawley (Indianapolis, Indiana, U.S.A.) at three weeks of age, andplaced on low salt diet (Lab Diet powder containing 0.4% NaCI, PM! Feed, Inc., St. Louis,MO, USA) or high salt diet (Lab Diet powder with extra 7.6% NaCI fine grain added andwell mixed - the final concentration of NaCI in high salt diet is 8%). Animals werepermitted to drink tap water ad libitum. After six weeks the rats were anaesthetized withhalothane and sacrificed by cervical dislocation (Sunga and Rabkin 1992). Tissues weretaken rapidly and hearts, trimmed of atria and main vessels, were weighed. Brain partswere further dissected on ice. All tissues were immediately snap-frozen in liquid nitrogenand stored at 140°C until further measurements.111.2. Isolation of Total RNA16Total RNA was extracted from tissues by the method of Chomczynski and Sacchi(Chomczynski and Sacchi 1986). For pituitary samples, RNA was extracted from two ratpituitary glands pooled together. Briefly, homogenization of tissues was carried out in 4M guanidine isothiocyanate, 25 mM trisodium citrate (pH 7.0), 0.5% sodium N-laurylsarcosine, and 0.1 M 2-mercaptoethanol. Sodium acetate (pH 4.0) was then addedand brought to 0.2 M. Protein in the homogenate was removed by phenol-chloroformextraction. RNA was precipitated in isopropanol and washed with 70% ethanol. Theprecipitated RNA was dissolved in diethyl pyrocarbonate-treated water and quantitatedby optical absorbance at 260 nm. Protein contamination was assessed by the ratio of260/280 nm absorbance. Samples with a ratio less than 1.8 were subject to furtherphenol-chloroform extraction. The ratios of absorbance at 260 nm to that at 280 nm insamples for further analysis were 1.8- Complementary DNA ProbesE. coli RR1 which harbours plasmid pBetagal containing 0.14 kb POMCcomplimentary DNA corresponding to the coding sequence for 0-endorphin and /3 -MSHwas supplied by American Type Culture (U. S. Patent No. 4,350,764). Because of thelarge size of original vector pBR322 (4.3 kb) and the relatively small size of the 0.14 kbcDNA, we failed to isolate the POMC cDNA fragment from pBetagal. So we cut pBetagalwith Eco RI and ligated the 0.14 kb cDNA fragment with a smaller plasmid vector pUC18(2.7 kb and also cut with Eco RI). The competent E. coli was then transformed with theligated plasmid and the transformed E. coli was cloned. The 0.14 kb POMC cDNA17fragment was successfully purified this way. The new plasmid with pUC18 vector and0.14 kb POMC cDNA is called pUCme with me implying nucleic acid sequencescorresponding to p-MSH and p-endorphin. E. coli C600, transformed with plasmidpYSEC1 containing 0.93 kb cDNA of preproenkephalin A, was a gift from Dr. Sabol ofNational Institute of Health, U.S.A. (Yashikawa et al. 1984). E. coli containing plasmid wasgrown overnight in LB medium with 100 ug/ml ampicillin and the plasmid was extracted.Plasmids pbetagal and pYSEC1 were then cut with restriction enzymes Eco RI and SacI-Sma 1 respectively and cDNA fragments were isolated by electrophoresis. A 1.25 kbbovine p-actin cDNA was also used as an internal standard (Gordon et al. 1992). ThecDNA probes were labelled with [32P]a-dCTP using the method of random oligonucleatideprimers.111.4. RNA Electrophoresis and Northern Blot AnalysisNorthern blot analyses were used to identify and quantitate messenger RNA forPOMC and preproenkephalin A. 5 - 20 gg total RNA samples were denatured in 50%formamide, 18% formaldehyde, 20 mM 3-(N-morpholino)-propane-sulphonic acid (MOPS),5 mM sodium acetate, 1 mM EDTA, pH 7.0; size-fractionized by electrophoresis through18% formaldehyde and 1% agarose gel, in 20 mM MOPS, 5 mM sodium acetate, 1 mMEDTA, pH 7.0; blotted onto Bio-Rad Zeta-Probe nylon membrane. The blots wereprehybridized at 65°C for 30 minutes in buffer containing 0.5 M NaH2PO4 (pH 7.2), 7%sodium dodecyl sulfate (SDS) and 1 mM EDTA and hybridized in 10 ml of renewedidentical buffer with about 106 cpm/ml phosphorus-32-labelled probe overnight. Blots18were then washed twice in 200 ml of solution containing 40 mM NaH2PO4 (pH 7.2), 5%SDS and 1 mM EDTA, and twice in 200 ml of solution containing 40 mM NaH2PO4 (pH7.2), 1% SDS and 1 mM EDTA. Each wash took place at 65°C for 15 minutes. Blotswere subsequently dried and exposed to Kodak X-OMAT x-ray films for 6-16 hours indouble intensifying screens at -70°C. The signals on exposed film were measured withBio-Rad Model 620 video densitometer (Bio-Rad Laboratory Inc., Hercules, California,U.S.A.).IV. MaterialsZeta-Probe nylon membrane and ultra pure DNA grade agarose were from Bio-RadLaboratories, Richmond, California, USA. Oligonucleotide primers (hexamers) werepurchased from Pharmacia LKB Biotechnology Inc., Piscataway, New Jersey, USA.Klenow fragment of DNA polymerase I and restriction endonucleases Eco RI, Sma I andSac I were from BRL Life Technologies, Inc., Gaithersburg, Maryland, USA. [3211-dCTPwere supplied by Amersham Canada Limited, Oakville, Ontario. Guanidine thiocyanate,unlabelled dATP, dGTP, and dTTP, and synthetic oligopeptides Met-enkephalin, Leu-enkephalin, MEAGL and MEAP, were the products of Sigma Chemical Company, St.Louis, Missouri, USA. All other chemicals used were provided by BDH Inc., Toronto,Ontario.RESULTSI.^Effects of intravenously administered enkephalins in the unanesthetized Sprague-Dawley ratsAt the doses of 3.6 and 36 nmoles, the four proenkephalin-derived peptides, LE, ME,MEAGL or MEAP, had no effect on heart rate (Figure 1) and arterial blood pressure(Figure 2). At the 360 nmoles dose, all the four peptides were found to briefly decreaseheart rate (Figure 1 and 3). Heart rate fell significantly 10 seconds after the intravenousadministration, and returned to the baseline level about 30 seconds after theadministration. The maximum fall in heart rate occurred at about 10 seconds afterintravenous administration of 360 nmole dose of LE, ME, MEAGL or MEAP (Figure 4).The maximum fall in heart rate after LE administration was -114 -± 20 (bpm ± SEM, p =0.002); ME, -108 ± 23 (p = 0.018); MEAGL, -111 ± 30 (p = 0.05); MEAP, -127 ± 33 (p= 0.019).Mild although not significant decreases in arterial pressure were observed at dosesof 360 nmoles (Figure 5). After intravenous administration of 360 nmole of eachenkephalin the maximum decreases in mean arterial pressure were (in mmHg): LE, -12± 8; -12 ± 10; MEAGL, -8 ± 7; MEAP, -23 ± 23. MEAP but not met-enkephalin, leu-enkephalin, or MEAGL had a delayed positive pressor effect on arterial blood pressure(Figure 5). The rise in mean arterial blood pressure started 20 seconds after intravenous19Figure 1. The effects of intravenous administration of proenkephalin-derived peptides onheart rate. The effects of each peptide were studied in separate animals (n =5 for LE andMEAP; n=4 for ME and MEAGL). Paired two-tailed t-test was used to compare heartrates after administration of enkephalins with respective baseline heart rate beforeadministration of the peptide concerned. Bars represent the means of maximum changein heart rate (bpm) ± SEM after different doses (nmole/rat) of enkephalins. Mean restingheart rate (bpm) ± SEM for each peptide: LE, 336 ± 9; ME, 327 ± 11; MEAGL, 348 ± 6;MEAP, 350 ± 7. p < 0.05 and p < 0.01 respectively, compared with respectivebaseline heart rate.20**Change in mean arterial pressure (mmHg)LE^ME^MEAGL^MEAPIIIII 3.6 nMole 36 nMole ^ 360 nMoleFigure 2. The effects of intravenous administration of proenkephalin-derived peptides onmean arterial blood pressure. The effects of each peptide were studied in separateanimals (n=5 for LE and MEAP; n=4 for ME and MEAGL). Paired two-tailed t-test wasused to compare mean arterial pressure after administration of enkephalins withrespective baseline mean arterial pressure before administration of the peptide concerned.Bars represent the averages of maximum change in mean arterial pressure (mmHg) ±SEM after different doses (nmole/rat) of peptides. Mean resting arterial pressure (mmHg)± SEM for each peptide: LE, 84± 4; ME, 82± 6; MEAGL, 85± 11; MEAP 88± 8. P <0.01, compared with respective baseline mean arterial pressure.21906030-30Heart rate (bpm)40030020010000^15^30^45^60^75^90 100Time (second)LE^ME ^ MEAGL^D^ MEAP^x^ NSFigure 3. Time course of heart rate after intravenous administration of 360 nmole peranimal of LE, ME, MEAGL, MEAP or NS (normal saline or 0.9% NaCI). Each peptide wasadministered to separate animals. The equal volume (0.5 ml) of normal saline to thevolume of enkephalin solution was administered to the animals before each peptideadministration to be used as controls.22906030Change in mean arterial pressure (mmHg)MEAPMEAGLLE^ME-60-30111110 sec   20 sec  30 sec 60 sec LJ 90 secFigure 5. The effects of intravenous administration of proenkephalin-derived peptides onmean arterial blood pressure. The effects of each peptide were studied in separateanimals (n=5 for LE and MEAP; n=4 for ME and MEAGL). Paired two-tailed t-test wasused to compare mean arterial pressure after administration of enkephalins withrespective baseline mean arterial pressure before administration of the peptide concerned.Bars represent means of change in mean arterial pressure (mmHg) ± SEM at differenttime after administration of 360 nmole/rat of each peptides. p < 0.05 and p < 0.01respectively, compared with respective baseline mean arterial pressure.2425administration of MEAP, immediately after the recovery of heart rate. The increase inmean arterial blood pressure was greatest at 25 seconds (59 -± 9 mmHg, p = 0.015) and30 seconds (56 -± 8, p = 0.002) after intravenous MEAP administration. The arterialblood pressure returned almost to the baseline level at 90 seconds after theadministration (12 -± 6 mmHg above the baseline level).II. Effects of enkephalins on isolated hearts of Sprague-Dawley ratsAt the concentration of 10-6 M in the perfusate, neither of the four proenkephalinproducts, LE, ME, MEAGL and MEAP, significantly altered the developed pressure of theleft ventricle (Figure 6). The maximum change in developed pressure of the left ventricle(mean ±: SEM in mmHg) during the perfusion of LE was 0.13 -± 1.42; ME, 1.50 -± 1.50;MEAGL, 1.25 ± 1.25; MEAP, -0.13 :It 1.42.III. Northern blotting analysis: decreased expression of POMC and increasedexpression of ppENK in S rats111.1. Ventricular hypertrophy in S ratsBoth R and S rats on the 8% NaCI diet had lower body weight than theircounterparts on the 0.4% NaCl diet. S rats on high salt diet had remarkable loss ofweight in the fifth and sixth week when they manifested the symptoms of heart failure (p< 0.01 compared with S rats on low salt diet or R rats on high salt diet). S rats on thehigh salt diet manifested the consequence of hypertension by developing obvious leftventricular hypertrophy after six weeks on high salt diet. Relatively less severe cardiachypertrophy also developed in S rats after six weeks on low salt diet. R rat group onLE ME L MEAGL MEAPFigure 6. Effects of different enkephalins on developed pressure in the left ventricle of theisolated heart. Shown here are maximal changes (mmHg) ± SEM in left ventricledeveloped pressure from the baseline levels after the isolated heart was perfused withsolution containing 10-6 M of LE, ME, MEAGL or MEAP. Each bar represents results fromeight experiments. Paired two-tailed t-tests were used and no significant change frombaseline in developed pressure was demonstrated after enkephalin perfusion. Baselinedeveloped pressure was 91 ± 15 mmHg.2627Table 1. Heart weight and body weight of Dahl salt-sensitive and salt-resistant rats aftersix weeks on high salt (8%) and low salt (0.4%) dietn^Body Wt. (g)^Heart Wt. (g)^Heart/Body (%)RH 16 312 ± 6 0.88 ± 0.02+ 0.28 ± 0.01+SH 16 257 ± 16** ++ 1.17 ± 0.05*** + 0.46 ± 0.02*** +++AL 10 320 ± 7 0.80 ± 0.03 0.25 ± 0.01SL 10 336 ± 15 1.03 ± 0.04*** 0.31 ± 0.02*Results represent means ± SEM. n = number of animals; RH, Dahl salt-resistant rats onhigh salt diet; SH, Dahl salt-sensitive rats on high salt diet; RL, Dahl salt-resistant rats onlow salt diet; SL, Dahl salt-sensitive rats on low salt diet. Student's unpaired (unequalvariance) t-tests were used in the statistical analysis; *p < 0.05, p < 0.01 and * p <0.001 respectively as compared with that of R rats on the same diet; +p < 0.05, ffp <0.01 and +41-13 < 0.001 respectively as compared with that of the same strain on low saltdiet.28high salt diet also had relatively larger hearts compared with R rats on low salt diet (Table1).111.2. Increased expression of ppENK mRNA in the cardiac ventricles of S ratsIn both R and S rats, the 1.5 kb ppENK messenger RNA was shown to be presentin different brain regions. The liver, kidney, and adrenal gland were not shown to haveany significant amount of ppENK mRNA and the larger ppENK mRNA of 1.75 kb wasabundantly expressed in the testicular tissues (Figure 7). There were no differences ofboth p-actin mRNA (2.1kb) and ppENK mRNA levels in different brain regions betweenR and S rats on 8% NaCI diet (Figure 8). The levels of ppENK mRNA in atria were aboutthe same. In contrast, there were dramatic increases in the amount of ppENK mRNA inboth left and right ventricles in S rats as compared to R rats whether the R and S ratswere on high or low salt diet (Figure 9 and 10). As comparison, the amount of p-actinmRNA was the same in both ventricles between the R and S rats (Figure 11). Theamount of ppENK mRNA in the left and right ventricles was more than doubled in S ratsthan in R rats on both high and low salt diet (Figure 12). In contrast, the hearts--fromwhich total RNA was extracted for Northern blot analysis of ppENK messenger RNA--notonly of S rats on high and low salt diet but also of R rats on high salt diet, developedventricular hypertrophy (Figure 13). There was also more ppENK mRNA in the right (p< 0.05) and left (p < 0.05) ventricles of S rats on high salt diet than S rats on low saltdiet. The expression of ppENK mRNA in both left and right ventricles of R rats on highsalt diet was the same as R rats on low salt diet. In both R and S rats, the level ofR CORTEXR DIENCEPHALONR BRAIN STEMR CEREBELLUMR TESTISR LIVERR KIDNEYR ADRENAL29Figure 7. Northern blot analysis of rat tissue total RNA hybridized to [32PHabeled ppENKand p -actin cDNAs. Each lane contains 20 A g of total RNA from tissues of R rats on highsalt (8%) diet. The size of ppENK mRNA is about 1.5 kb and p -actin mRNA is about 2.1kb. Exposure, 16 hours with Kodak AR film with double intensifying screens. R, Dahlsalt-resistant rat. The tissues include: cortex (cerebral cortex), diencephalon (thalamusplus hypothalamus), brain stem (midbrain plus pons plus medulla oblongata), cerebellum,testis, liver, kidney, and adrenal gland.R CORTEXS CORTEXR DIENCEPHALONS DIENCEPHALONR BRAIN STEMS BRAIN STEMR CEREBELLUMS CEREBELLUM30Figure 8. Northern blot analysis of total RNA hybridized to [3211-labeled ppENK and /3-actin cDNAs. Each lane contains 20 g g of total RNA from tissues of different brainregions of R or S rats on high salt (8%) or low salt (0.4%) diet. The size of ppENK mRNAis about 1.5 kb and /3-actin mRNA is about 2.1 kb. Exposure, 16 hours with Kodak ARfilm with double intensifying screens. R, Dahl salt-resistant rat; S, Dahl salt-resistant rat.Brain regions include cortex (cerebral cortex), diencephalon (thalamus plushypothalamus), brain stem (midbrain plus pons plus medulla oblongata), and cerebellum.R R. VENTRICLES R. VENTRICLER L. VENTRICLES L. VENTRICLER ATRIAS ATRIAFigure 9. Northern blot analysis of total RNA hybridized to [321=]-labeled ppENK cDNA.Each lane contains 20 1.1 g of total RNA from cardiac tissues of R or S rats on high salt(8%) diet. Exposure, 16 hours with Kodak AR film with double intensifying screens. R,Dahl salt-resistant rat; S, Dahl salt-resistant rat. The heart was dissected into: atria (leftatrium plus right atrium), right (R.) ventricle, and left (L.) ventricle.R R. VENTRICLES R. VENTRICLER L. VENTRICLES L. VENTRICLER ATRIAS ATRIAFigure 10. Northern blot analysis of total RNA hybridized to [321'1-labeled ppENK cDNA.Each lane contains 20 A g of total RNA from cardiac tissues of R or S rats on low salt(0.4%) diet. Exposure, 16 hours with Kodak AR film with double intensifying screens. R,Dahl salt-resistant rat; S, Dahl salt-resistant rat. The heart was dissected into: atria (leftatrium plus right atrium), right (R.) ventricle, and left (L.) ventricle.33R R. VENTRICLES R. VENTRICLER L. VENTRICLES L. VENTRICLER ATRIAS ATRIAFigure 11. Northern blot analysis of total RNA hybridized to [32P]-labeled 3 -actin cDNA.Each lane contains 20 p, g of total RNA from cardiac tissues of R or S rats on high salt(8%) or low salt (0.4%) diet. Exposure, 16 hours with Kodak AR film with doubleintensifying screens. R, Dahl salt-resistant rat; S, Dahl salt-resistant rat. The heart wasdissected into: atria (left atrium plus right atrium), right (R.) ventricle, and left (L.) ventricle.34Figure 12. Northern blot densitometric determinations of cardiac ppENK messenger RNAlevels of Dahl R and S rats on 8% and 0.4% NaCI diet. Each bar represents results ofdensitometric determination of ppENK messenger RNA signals from four animals (mean± SEM). LV, left ventricle; RV, right ventricle; RH, Dahl salt-resistant rats on high salt diet;SH, Dahl salt-sensitive rats on high salt diet; RL, Dahl salt-resistant rats on low salt diet;SL, Dahl salt-sensitive rats on low salt diet. Student's unpaired two-tailed **(equal orunequal variance) t-tests were used in the statistical analysis; p < 0.05 and p < 0.01respectively, compared with that of R rats on the same diet; +p < 0.05, compared withthat of S rats on low salt diet.Figure 13. Ratio of heart weight and body weight of animals from which cardiac ppENKmRNA was determined by Northern blot analysis and densitometry (see Figure 12).Animals were Dahl salt-sensitive and salt-resistant rats after six weeks on high salt (8%)or low salt (0.4%) diet. Each bar represents means from four animals (%) ± SEM. RH,Dahl salt-resistant rats on high salt diet; SH, Dahl salt-sensitive rats on high salt diet; RL,Dahl salt-resistant rats on low salt diet; SL, Dahl salt-sensitive rats on low salt diet.Student's unpaired* two-tailed (equal or unequal variance) t-tests were used in thestatistical analysis; p < 0.05 and p < 0.01 respectively, compared with that of R ratson the same diet; ++p < 0.01, compared with that of the same strain on low salt diet.3536ppENK mRNA was much higher in the left ventricle than in the right ventricle.111.3. Increased expression of POMC messenger RNA in R rats on high salt dietThe 1.1 kb POMC messenger RNA was abundant in pituitary tissues. It was notreadily detectable in liver, heart, kidney, adrenal gland, or other parts of the brain (Figure14). There was more POMC mRNA in the pituitary of R rats relative to S rats after 6weeks on the high salt group (Figure 15). In contrast, there were no differences inppENK and p-actin mRNA in the pituitary between R and S rats on high salt diet. Theamount of POMC mRNA was significantly (p < 0.01) greater and indeed two foldgreater in the pituitary of R rats than in the pituitary of S rats (Figure 16). This differencein the POMC messenger RNA levels in the pituitary between R and S was not detectedwhen these rats were put on a low salt diet (Figure 17). The levels of pituitary POMCmRNA in R rats on low salt diet were similar to that of S rats on high salt diet (Figure 18).S pituitaryR pituitaryR hypothalamusR cerebellumR brain stemR diencephalonR striatumR hippocampusR cerebral cortexR adrenalR liverR kidney37Figure 14. Northern blot analysis of rat tissue total RNA hybridized to [32P]-Iabeled POMCcDNA. Each lane contains 5 lig of total RNA from pituitary or 20 lig of total RNA fromother tissues of rats on high salt (8%) diet. Exposure, 16 hours with Kodak AR film withdouble intensifying screens. R, Dahl salt-resistant rat; S, Dahl salt-sensitive rat. Othertissues include: hypothalamus, cerebellum, brain stem (midbrain plus pons plus medullaoblongata), diencephalon (thalamus), striatum, hippocampus, cerebral cortex, adrenalgland, and kidney.Figure 15. Northern blot analysis of rat pituitary total RNA hybridized to [32P]-labeledcDNA for POMC, or preproenkephalin A (proENK), or p -actin. Each lane contains 5 A gof total RNA from pituitary of R and S rats on high salt (8%) diet. Exposure, 6 hours forPOMC determination and 16 hours for preproenkephalin A and p -actin analysis withKodak AR film with double intensifying screen. R, Dahl salt-resistant rat; S, Dahl salt-sensitive rat; p -actin on left, ppENK on middle, and POMC on right.38Arbitrary absorbance units543210POMC^ppENK^ie -actinMel RH M SHFigure 16. Northern blot densitometric determinations of messenger RNA levels in thepituitary of rats on high salt (8%) diet. Data were from six experiments and eachexperiment used RNA samples extracted from two rat pituitary glands. Ordinate isarbitrary densitometric absorbance units (mean ± SEM) of signals in Northern blotautoradiography and the abscissa is POMC m RNA (6 hours exposure), preproenkephalinA (ppENK) mRNA (16 hours exposure), and 13 -actin mRNA (16 hours exposure). RH,Dahl salt-resistant rats on high salt diet; SH, Dahl salt-sensitive rats on high salt diet.Student's unpaired two-tailed (equal or unequal variance) t-tests were used in thestatistical analysis; p < 0.01 compared with RH.3940RLSLRHSHRLSLRHSHFigure 17. Northern blot analysis of rat pituitary total RNA hybridized to [32P]-labeledPOMC. Each lane contains 5 A g of pituitary total RNA. Exposure, 6 hours with KodakAR film with double intensifying screens. RH, Dahl salt-resistant rats on high salt diet; SH,Dahl salt-sensitive rats on high salt diet; RL, Dahl salt-resistant rats on low salt diet; SL,Dahl salt-sensitive rats on low salt diet.Figure 18. Northern blot densitometric determinations of pituitary POMC messenger RNAlevels of Dahl R and S rats on 8% NaCI diet (n = 6 experiments) or 0.4% NaCl diet (n =3 experiments). Results represent means ± SEM of densitometric absorbance fromsignals on Northern blot autoradiography; RH, Dahl salt-resistant rats on high salt diet;SH, Dahl salt-sensitive rats on high salt diet; AL, Dahl salt-resistant rats on low salt diet;SL, Dahl salt-sensitive rats on low salt diet. Student's unpaired two-tailed (equal orunequal variance) t-tests were used in the statistical analysis; p < 0.01, compared withS on the same diet; +p < 0.05, compared with R on low salt diet.42DISCUSSIONWith the development of molecular biology, ligand receptor assay, and otherbiochemical techniques, the knowledge on opioid peptides is expanding rapidly withinterest coming from both scientists and clinicians. The interactions of different opioidpeptides and receptors and their relations with other hormones, transmitters, andreceptors are complex. These peptides and receptors have a wide distribution in varioustissues. More than a dozen endogenous opioid peptides have been identified within thebody (Kitchen 1985). At least five types opioid receptors have been defined and someof them have been subtyped, and more opioid receptor types and subtypes need furtherclarification (Kitchen 1985; Pasternack 1993; Quirion 1992; Rabkin and Redston 1987).The mechanisms of regulation in biosynthesis and release of these peptides have notbeen completely understood and how they affect the target cells is largely unknown. Dueto the fact that opioid peptides are involved in the modulation of such diverse functionsas pain perception, reproduction, circulation, and behaviour, and that different opioidsand receptors have various and even opposite effects on the same target cell, thefunction of each peptide has to be considered individually.The roles of endogenous opioids in the regulation of blood pressure and in thepathogenesis of hypertension have not been completely defined. Exogenous opioidpeptides induce different effects on blood pressure and heart rate depending on theroute of administration and on the animal model involved (Feuerstein and Siren 1987).The sites of action in the cardiovascular system are not clear. The investigation of43changes in endogenous opioids and their receptors in hypertensives has not beenpursued vigorously. Because of the complexity of the opioid system and cardiovascularregulation, their relationship is still a puzzle. The results in this study suggest the rolesof endorphins and enkephalins in the regulation of the cardiovascular system and indicatethat decreased endorphins and increased enkephalins may be closely involved in thepathogenesis of hypertension.I. Effects of intravenous administration of enkephalinsIntravenous administration of enkephalins induce different effects on blood pressureand heart rate depending on animal species and anaesthesia. In urethane-anaesthetizedSprague-Dawley rats, Met-enkephalin and Leu-enkephalin decreased heart rate and bloodpressure (Wei et al. 1980). In pentobarbital-anaesthetized mongrel cats, Leu-enkephalininduced a transient rise and a more prolonged decline in mean arterial blood pressurewhile Met-enkephalin caused only a decline in mean arterial pressure, and neitherenkephalin significantly altered heart rate (Moore and Dowling 1980). In consciousnormotensive Wistar-Kyoto and spontaneously hypertensive rats, intravenousadministration of Leu-enkephalin briefly increased blood pressure and heart rate (Schazet al. 1980). When administered intravenously, enkephalins decreased arterial bloodpressure and heart rate in pentobarbital-anaesthetized dogs, and increased bloodpressure and heart rate in conscious dogs (Sander et al. 1981, 1982). In a more recentstudy, intravenously administered Met-enkephalin, Leu-enkephalin, MEAGL and MEAPproduced vasodepression and bradycardia in urethane-anaesthetized rats (Douglas and44Kitchen 1992). In the same study, tachycardia and pressor effects were also observedimmediately following vasodepression and bradycardia in the MEAP administration. Inconscious human subjects, methionine-enkephalin increases arterial blood pressure andheart rate (Giles et al. 1987). From all these data available, it seems that enkephalinsproduce mainly pressor and tachycardia effects in conscious animals and human beingsand vasodepression and bradycardia effects in the anaesthetized animals. Our data inconscious animals show extremely brief inhibitory effects of enkephalins on heart rate anda longer pressor effect by MEAP on arterial blood pressure. We chose the SpragueDawley rat because Dahl rats were selectively bred from the Sprague Dawley rat.Importantly, their use eliminated potential changes in opioid receptor number and affinitythat may occur with development of hypertension (Dumont and Lemaire 1988). Our studyused bolus injections of opioids to observe the pattern of the response. Because of thecomplexity of pharmacokinetic and pharmacodynamic mechanisms of intravenouslyadministered enkephalins, the mechanisms involved in the effects of enkephalins on heartrate and of MEAP on blood pressure needs to be investigated by other approaches aswell. MEAP produced the most definite pressor response. The dipeptide Arg-Phe derivedfrom the hydrolysis of MEAP was believed to be responsible for the hypertensive effectof MEAP (Douglas and Kitchen 1992).II.^The effect of enkephalins on the isolated heartAvailable data concerning the direct effect of enkephalins on the heart has beenscarce and conflicting, but the presence of opioid receptors and small amount of45bioactive enkephalins suggests that enkephalins may regulate cardiac function directly(Dumont and Lemaire 1988; Dumont et al. 1991; Krumins 1987; Mantelli et al. 1987; Weiheet al. 1985). Enkephalins were shown to decrease the chronotropic response of isolatedspontaneously beating rat atria to adrenergic agonists (Eiden and Ruth 1982). Thereduction in catecholamine-induced tachycardia was related to inhibition of Ca2+accumulation by the rat atria (Ruth et al. 1983). In the spontaneously beating guinea pigatria, however, leu-enkephalin augmented the chronotropic response to norepinephrineaccompanied by a naloxone reversible increment in Ca2+ uptake by atrial preparation(Ruth et al. 1984). In the isolated cells, enkephalins increased cyclic AMP content,calcium uptake, and contractility in cultured chick embryo heart cells (Laurent et al. 1986).But in a recent study, enkephalins were shown to increase inositol 1,4,5-triphosphate inthe cardiomyocyte and to cause Ca2+ release and depletion from intracellular store sites,which results in gradually reduced contractility (Ventura et al. 1992). ME and LE werealso shown to be able to inhibit ouabain-sensitive Na+-K+ ATPase and Ca2+-dependentATPase activities in bovine cardiac sarcolemma (Ventura et al. 1987). The inhibition ofCa2+-dependent ATPase and/or Na+-K+ ATPase activities of the sarcolemma are relatedto the cardiodepression (Dhalla et al. 1976, 1977, 1978). But the inhibition of sarcolemmalNa+-K+ ATPase activity is also believed to contribute to the positive inotropic effect ofcardiac glycosides (Schwartz et al. 1975). Another interesting but contradictory reportwas that opioid receptor stimulation increased cytosolic pH and myofilamentresponsiveness to Ca2+ in cardiac myocyte, most likely via protein kinase C activation ofamiloride-sensitive Na+/H+ exchanger (Ventura et al. 1991). Although evidence in direct46cardiac effects of enkephalins seems to be conflicting, the present study did not find anyinotropic effects of these enkephalins in the isolated heart. The concentrations used, 10-8and 1(16 M, were relatively large considering the doses that produced bradycardia andhypertension in the intact animal. Our study, which demonstrated increased expressionof enkephalins in the hypertrophic ventricles, as well as other reports that show increasedexpression of enkephalins in the cardiomyopathic ventricles (Ouellette and Brakier-Gingras 1988) and in the freshly infarcted ventricles (Paradis et al. 1992), suggestpossible roles of enkephalins in the modification of cardiomyocyte structure and/or theregulation of cardiac function under some circumstances (Rabkin 1992), but not an actiondirectly on cardiac contractility.III. Transcription of POMC and ppENK genes in different tissues111.1. Messenger RNA for ppENKPreproenkephalin mRNA has been found in various tissues such as brain, spinalcord, adrenal medulla, gastrointestinal tract, and heart (Hoegler et al. 1989; Howells etal. 1986; Jingami et al. 1984). A longer transcript was also reported to exist in testis(Garrett et al. 1989). We confirmed the presence of ppENK mRNA in testis, brain andheart, but failed to demonstrate it in the adrenal gland. The reason may be that we usedthe whole adrenal gland for total RNA extraction while other investigators specifiedadrenal medulla. The expression of ppENK in the cardiac ventricle increases with ageof the animals (Dumont et al. 1991; Springhorn and Claycomb 1989). We found thatppENK mRNA concentrations differed among the parts of the heart. In both R and S47rats, there was more ppENK messenger RNA in the left ventricle than in the right ventriclewhich contained more ppENK mRNA than atria. In the adult Dahl R rats, the ventricleswere at least as rich in ppENK mRNA as the brain, while in adult S rats, the ventriclescontain several times more ppENK mRNA than brain. Enkephalins and precursor peptidehave been reported to be much lower in the heart (Dumont et al. 1991; Howells et al.1986). The discrepancy between ppENK mRNA and enkephalin peptides in the cardiacventricles may lie in the fact that ventricular cardiomyocytes do not store the peptides(Bloch et al. 1986). It has been demonstrated that ppENK transcript is actively translatedinto polypeptide (Low et al. 1990). But the abundance of mRNA may also means thatthere exists the translational control of protein synthesis in the heart (Howells et al. 1986).It is understandable that this control mechanism gives a faster response in a situation inwhich there is a need for the peptide.111.2. Messenger RNA for POMCPOMC mRNA was reported to be present in small amount in hypothalamus,amygdala, cerebral cortex, adrenal medulla, thyroid, thymus, duodenum, lung, testis,ovary and placenta, in addition to its abundance in pituitary tissues (Chen et al. 1984,1986; Civelli et al. 1982). POMC transcripts detected in these tissues were found to beshorter than in the pituitary while a longer transcript was also observed in hypothalamus(Chen et al. 1984, 1986; Civelli et al. 1982; Jeannotte et al. 1987; Jingami et al. 1984). Inour study, POMC messenger RNA was found in very high concentration in pituitarytissues. The explanation for our failure to demonstrate POMC mRNA in hypothalamus,48cerebral cortex and testis may lie in that only very small amount of POMC mRNA wasfound in these studies even though poly(A) RNA was used in Northern blot analysis(Civelli et al. 1982; Jeannotte et al. 1987). We did not resort to poly(A) RNA method inour study.IV. Differences in the expression of POMC and ppENK in R and S ratsIV.1. Increased ppENK mRNA in the cardiac ventricular tissues of S ratsEnkephalins have been the most investigated among endogenous opioid peptidesin the regulation of blood pressure and the pathogenesis of hypertension. Microinjectionof leu- and met- enkephalin into certain areas in the brain can profoundly alter bloodpressure (Feuerstein 1985; Rabkin 1989). These enkephalins may also alter arterial bloodpressure and heart rate when administered systemically (Moore and Dowling 1980;Sander et al. 1981; Simon et al. 1978; Wei et al. 1980). The levels of leu-enkephalin inthe pituitary gland and several brain areas are substantially different in spontaneouslyhypertensive rats (SHR) as compared with normotensive animals (Feuerstein et al. 1983).Preproenkephalin A (ppENK) messenger RNA levels were found increased inhypothalamus, midbrain, cerebellum, and thoracic cord and decreased in pons-medullaof SHR relative to the normal controls (Hoegler et al. 1989). Increased ppENK mRNA wasalso observed recently in the heart of SHR as compared with normotensive controlanimals (Dumont et al. 1991). Opioid peptides were also shown to have a greater pressorresponse in SHR than in the normotensive controls (Rockhold et al. 1981; Schaz et al.1980; Yukimura et al. 1981). But there is comparatively much less data available about49enkephalins in models of hypertension other than the SHR.We found increased ppENK transcription in the Dahl S rats no matter whether theywere on high or low salt diets. The differences in the ppENK mRNA concentrationbetween R and S rats were found to be limited to the ventricles. Enkephalins producedin the heart may act as local chemical mediators to regulate the function ofcardiomyocytes; they may be transported to target tissues through the blood stream towork as hormones. The presence of opioid receptors and small amounts of bioactiveenkephalins suggests at least some local functions (Dumont and Lemaire 1988; Dumontet al. 1991; Krumins 1987; Mantelli et al. 1987; Weihe et al. 1985). Enkephalins increasedcyclic AMP content, calcium uptake, and contractility in cultured chick embryo heart cells(Laurent et al. 1986). But in a recent study, enkephalins were shown to increase inositol1,4,5-triphosphate in the cardiomyocyte and to cause Ca2+ release and depletion fromintracellular store sites, which results in gradually reduced contractility (Ventura et al.1992). Met- and Leu-enkephalin were also shown to be able to inhibit ouabain-sensitiveNa+-K+ ATPase in bovine cardiac sarcolemma (Ventura et al. 1987). Depression of thisenzyme has been reported in the failing hearts (Dhalla et al. 1976). Another interestingdiscovery is that opioid receptor stimulation has been found to increase cytosolic pH andmyofilament responsiveness to Ca2+ in cardiac myocyte, most likely via protein kinase Cactivation of amiloride-sensitive Na+/H+ exchanger (Ventura et al. 1991). The regulationof cell pH in turn is intimately involved with the control of cell growth and proliferation(Pouyssegur 1985; Vallega et al. 1988).The transcription of the ppENK mRNA is regulated by second messengers linked50to the signalling pathways of the sympathoadrenergic system. Indeed, various studieshave demonstrated that the ppENK gene is regulated at the transcription level by cyclicAMP through activation of cAMP-dependent protein kinase A and (or) via the activationof protein kinase C by phorbol esters (Chu et al. 1991; Comb et al. 1986; Springhorn andClaycomb 1989). Other studies have shown that the concentration of intracellular Ca2+also plays an important role in the regulation of ppENK gene expression (Kley 1988; Kleyet al. 1987). The increased ppENK mRNA in the ventricular tissue has been reported inthe hypertrophic, cardiomyopathic, and infarcted hearts (Dumont et al. 1991; Ouelletteand Brakier-Gingras 1988; Paradis et al. 1992). In our study, S rats developed cardiachypertrophy whether on high or low salt diets, which is in conformity with otherobservations that S rats developed hypertension whether on high or low salt diets (Rapp1984). R rats on the high salt diet had relatively larger hearts which may be the result oflong-term volume overload. But only S rats on both diets were shown to have increasedventricular ppENK mRNA. The possible explanations for the increase in ventricularppENK includes that increased enkephalin expression in these heart conditions may bethe result of overstimulation by the sympathoadrenergic system and be the mediatorresponsible for cardiac hypertrophy (Springhorn and Claycomb 1989).It is also possible that enkephalins produced in the heart regulate blood pressurein other ways besides the direct cardiac action. The enkephalins in the plasma areextremely short-lived just as other bioactive peptides (Dupont et al. 1977; Meek et al.1977). Because opioid peptides in the heart are mostly in the form of precursor (Dumontet al. 1991; Howells et al. 1986), they may be released as precursor peptides and51converted into bioactive enkephalins by endothelial peptidases in a similar fashion thatangiotensinogen is cleaved into active angiotensins. The search for evidence on thisspecific peptidase has not been pursued, but active forms of enkephalin do exist in theplasma (Clement-Jones et al. 1980). Proenkephalins are mainly pressor peptides (Gileset al. 1987; Sander et al. 1981, 1982, 1989; Schaz et al. 1980). Our study, and work byDouglas and Kitchen (Douglas and Kitchen 1992), has shown that the proenkephalinproduct MEAP is the strong candidate as pressor mediator in the development ofhypertension. Increased production of MEAP and possibly other enkephalins in thecardiac ventricles may contribute to the initiation and/or maintenance of hypertension.We also found more ppENK messenger RNA in the ventricles of S rats on high salt dietcompared with S rats on low salt diet. The explanation may be that high salt intake willexacerbate the hypertension by further stimulating the production and release ofenkephalins from the heart.The mechanisms by which MEAP and other enkephalins increase blood pressureare not completely understood. Enkephalins apparently do not cross the blood-brainbarrier in significant quantities (Pardridge and Mietus 1981). Studies have demonstratedthe impairment in arterial and cardiac baroreflex in Dahl S rats (Gorden et al. 1981; Market al. 1987; Miyajima and Bunag 1987). One explanation for this is that Dahl S rats mightrelease more of a desensitizing humeral factor. Enkephalins were shown to be able tomodify baroreflex by attenuating Benzold-Jarisch reflex (Sander et al. 1989). Thus,increased production and release of enkephalins from the cardiac ventricles can elevateblood pressure by resetting cardiac and/or arterial baroreflex to a higher threshold.52Opiate compounds have also been shown to directly contract rat aortic strips (Lee andBerkowitz 1976). In cardiac myocytes opioid receptor stimulation has been found toincrease cytosolic pH which promotes cell growth and proliferation, and increasesmyofilament responsiveness to Ca2+ (Ventura et al. 1991; Pouyssegur 1985; Vallega etal. 1988). Whether opioid peptides have a similar effect on vascular smooth muscle cellsrequires examination.IV.2. Increased POMC mRNA in the pituitary of R rats on high salt diet: responsible forsalt resistance?Investigations of the cardiovascular effects of opioid peptides have identified that p-endorphin had inhibitory effects on blood pressure regulation. Decreased arterial bloodpressure was observed when p-endorphin was administered intravenously or centrally(Lemaire et al. 1978; Petty et al. 1982a). /3-Endorphin was decreased in plasma frompatients with primary hypertension, and reduction of blood pressure in these patients bycentral a2-agonists was accompanied by the return to normal level of 3-endorphin (Kraftet al. 1987, 1988). The antihypertensive agent clonidine increased the release of f3-endorphin from brain slices of SHR (Kunos et al. 1981). The antihypertensive andbradycardiac effects of clonidine and a-methyldopa were blunted by opiate antagonistsand antiserum to p-endorphin (Farsang and Kunos 1979; Farsang et al. 1980; Ramirez-Gonzalez et al. 1983). Thus p-endorphin is linked with reduction of blood pressure.The measurement of POMC mRNA provides a reflection of relative transcription rate,the usual rate-limiting step for peptide biosynthesis (Douglass et al. 1984). fl-Endorphin,53derived from its polypeptide precursor POMC in pituitary, is released into the bloodstream for transferring to the target tissues (Smith and Funder 1988). In this study, wefound decreased POMC mRNA in the pituitary of S relative to R. This points out thatreduced release of p-endorphin from the pituitary may be responsible for hypertensionin Dahl S rats under salt stress.POMC is also the precursor for ACTH, but it is unlikely that ACTH would account forthe effects as ACTH and cortisol increase blood pressure, so that if reduced POMCmRNA was translated into reduced ACTH that should lead to lower blood pressures,while high blood pressure is uniformly observed in Dahl S rat on high salt diet (Dahl etal. 1968).The search for the cause of salt-dependent hypertension has been the "holy grail"of many investigators in this field with many proposed explanations, including renal andneurohumeral mediators. The data in the present study suggests that failure to producesufficient /3-endorphin, which is a hypotensive peptide, contributes to the development ofhypertension in high salt state, as a salt resistant strain of animals increases its mRNA for13-endorphin precursor in response to a high salt diet. In contrast, the salt sensitive strain,which has similar amounts of mRNA in low salt state, does not increase the pituitaryPOMC response in the presence of a high salt load. This finding may characterize saltdependent hypertension because in another experimental hypertension study the SHR,pituitary concentration of 13 -endorphin has been reported to be increased (Bhargava etal. 1988; Gaida et al. 1985).Salt sensitivity has been reported in as much as fifty to sixty percent of human54hypertensive subjects (Dahl 1963; Weinberger et al. 1986) and especially morepredominant in Afro-Americans with hypertension (Luft et al 1979, 1985). Low renin levelswere observed in Dahl S rats (lwai et al. 1973; Rapp et al. 1978) and have also been oneof the predominant features of Afro-Americans with hypertension (Freis et al. 1983).Interestingly, in response to stress, Afro-American patients with hypertension showedsignificantly less release of 0-endorphin compared to normotensives (McNeilly andZeichner 1989). Considering all these observations together, it is reasonable to considerthat insufficient secretion of 0-endorphin may be specific to salt dependent hypertension.V. Summary1. Intravenous administration of proenkephalin-derived peptides Leu5-enkephalin (LE), Met5-enkephalin (ME), Met5-enkephalin-Arg6-G1y7-Leu8(MEAGL) and Met5-enkephalin-Ard-Phe7 (MEAP) briefly decrease heart rateand arterial pressure in conscious Sprague-Dawley rats; MEAP but not LE, MEor MEAGL can increase blood pressure when administered intravenously.2. These enkephalins have no direct effect on the ventricular contractility inisolated heart of Sprague-Dawley rat.3. ppENK messenger RNA is present in brain, testis, cardiac atria andventricles of Dahl rats.4. POMC messenger RNA is mainly expressed in the pituitary of Dahl rats.5. Greater amount of ppENK messenger RNA is present in the cardiacventricles of the S rats. High salt intake further increases the production ofppENK mRNA in the cardiac ventricles.6. Inadequate production of POMC mRNA was found in the pituitary of DahlS compared to Dahl R rat on high salt diets.7. These observations suggest the following hypothesis. In the Dahl saltsensitive rat on a high salt diet, there is an increased cardiac ppEnk leadingto increased production of MEAP which further increases blood pressure. Inthe Dahl salt sensitive rat on a low salt diet, there is a smaller increase incardiac ppENK reflecting its sensitivity to even 0.4% dietary NaCI. In additionthere is a limited production of POMC, the precursor of the vasodilator peptide/3-endorphin in S rats, hat may contribute to the development of hypertension,as R rats on high salt diet increase the expression of POMC and thus therelease of 0-endorphin from the pituitary (Figure 19).55+Blood pressure t<-- -- Cardiac ppENK (MEAP) t1Cardiac ppENK (MEAP),tr,^Cardiac ppENK (MEAP) t t:- - -si^,c 4'.,^+4- Blood pressure t^+,-, ,^,^t Blood pressure t tft_ i ^Blood volume t^.^, —. i.^, .^,Pituitary POMC (p -endorphin) ■-■Figure 19. Schematic summary of this work: Dahl S rat has an enhanced expression ofppENK and consequently bioactive peptide MEAP, which increases blood pressure.Under high sodium stress, Dahl R rat increases the expression of POMC and the releaseof f3 -endorphin from the pituitary. p -Endorphin is known to decrease blood pressurewhich is high in the case of high salt intake and increased blood volume. 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