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The sympathetic tone of the splanchnic nerve was quantified in renal and deoxycorticosterone (DOC) hypertensive rats under pentobarbital anesthesia by measuring equivalent stimulation frequency (ESF). ESF is the frequency of stimulus to the peripheral cut end of the nerve required to restore the preseverance level of arterial pressure. It is assumed to represent the average discharge rate of the nerve before severance. ESF was markedly increased in DOC hypertension 2 weeks after initiation of the DOC-salt treatment. It was relatively decreased after 10 weeks, but was still higher than in the controls. In renal hypertension (clipping of the left renal artery), ESF was decreased considerably 2 weeks and slightly 10 weeks after clipping. When the contralateral kidney had been removed simultaneously with clipping (clip plus uninephrectomy, ESF was not significantly changed. It is suggested that the sympathetic nervous system participates greatly in the pathogenesis of DOC hypertension but not of renal hypertension.

Although the mechanism of antihypertensive action of beta-adrenergic blocking drugs (beta-blockers) is not known, a theoretical advantage of cardioselective beta-blockers over nonselective ones has been proposed in the treatment of hypertension. To study this hypothesis, we examined cardiovascular responses to handling stress in spontaneously hypertensive (SHR) rats after a single (100 mg/kg) and multiple oral treatments (100 mg/kg per day for 17 d) with either atenolol or propranolol. Atenolol and propranolol markedly suppressed the tachycardia induced by handling stress after acute and chronic administration. Resting mean arterial pressure (MAP) was reduced by acute and chronic atenolol treatment, but not by propranolol. Stress-induced increase in MAP was significantly reduced by chronic treatment with propranolol, whereas no consistent effects were observed with atenolol. Acute treatment with guanethidine (30 mg/kg) markedly reduced the rise in MAP induced by stress. These results suggest that suppression of cardiac function by beta-blockers does not always attenuate the rise in MAP induced by stress, thus cardioselective beta-blockers might not confer any further reduction of the blood pressure increase due to sympatho-adrenal excitation. Inhibition of stress-induced MAP rise by propranolol could be mediated by a modulation of the catecholamine release.

A hypothesis that the beta-adrenergic blocking drugs may possess weak alpha-adrenergic blocking activity, and that their antihypertensive effect may be due to this combined alpha - and beta-adrenergic blockade was tested. Phenoxybenzamine (PBZ) and propranolol (PPL) were selected as alpha - and beta-blocking drugs, respectively. They were given orally at doses of 1--100 mg/kg per day, 5 days per week. Their combined antihypertensive effects were determined in spontaneously hypertensive; deoxycorticosterone and salt hypertensive; and two kidney, one clip renal hypertensive rats. PBZ treatment showed antihypertensive effect in three models of hypertensive rats. However, addition of PPL either did not affect or rather antagonized the effect of PBZ. Moreover, the combined treatment increased side-effects, typically seen as urinary incontinence. The results did not support the above hypothesis, at least when the blockade was caused with PBZ and PPL.

The structural organization of calmodulin genes in the spontaneously hypertensive rat (SHR) was extensively studied to search for alterations in calmodulin. We constructed genomic libraries of SHR and cloned all calmodulin-related genes in the genome. We also cloned and sequenced calmodulin complementary (c)DNA from a rat brain (Sprague-Dawley) cDNA library. We cloned three distinct calmodulin genes, naming them CaM I, II and III. Three distinct cDNA clones corresponding to these genes (pRCM1, pRCM3 and pRCM4) were also cloned. These SHR calmodulin genes all encoded normal calmodulin, and no alteration was found. Four processed pseudogenes, lambda SC9 for CaM I gene and lambda SC8, lambda SC19 and lambda SC27 genes for CaM II genes were also cloned and analysed.

Crude bullfrog angiotensin was prepared by modified Boucher's procedures. The kidney extract was incubated in vitro with homologous plasma. Crude angiotension, 15 micrograms equivalent to [Asp1, Ile5]angiotensin II in rat vasopressor assay, was purified further by three steps by SEP-PAK C18 cartridge, SP-Sephadex column chromatography, and high-performance liquid chromatography on Finepak SIL C18 column. Amino acid analysis was done on 64 ng of the purified material after acid hydrolysis. The fluorescent peptide mapping technique was used to confirm the amino acid sequence. It is proposed that bullfrog angiotensin is [Asp1, Val5, Asn9]angiotensin I.

To investigate localization of the action on blood pressure in the peptide chain of parathyroid hormone (PTH), we determined the effect of seven fragments of human (h) PTH-(1-34) fragments on blood pressure in anesthetized rats. hPTH-(1-7) and -(28-34) fragments had no effect up to 1 X 10(-6) mole/kg. hPTH-(8-34), -(13-34), -(18-34), and -(23-34) fragments showed significant hypotensive activity. The decrease in the chain length lowered the hypotensive activity. The results indicate that some amino acids in position 23-34 are necessary for the hypotensive activity. This accords with the previous report suggesting that 5 amino acids in position 24-28 are essential. In addition hPTH-(8-34) and -(13-34) fragments showed initial transient hypertensive activities. hPTH-(1-13) fragment had only hypertensive action, suggesting that some amino acids in position 8-13 are necessary for hypertensive activity.

We have devised a simple method to determine the ratios of cardiac to vascular beta-receptor blockage (C:V ratios) in normotensive rats anesthetized with pentobarbital, and applied it to six beta-adrenergic blocking drugs (beta-blockers). Mean blood pressure (BP) and heart rate (HR) were recorded. Isoproterenol (3 ng-100 microgram/kg, i.v.) was cumulatively injected before and after the intravenous administration of beta-blockers at two dose-levels. Thus shifts of the dose-response curves in BP and HR were observed. "In vivo pA2" values were calculated in molar doses per kg by Schild plots. The C:V ratio was calculated as antilogarithm of the difference of the mean in vivo pA2 value in HR from that in BP. Those of atenolol, bometolol, bunitrolol, pindolol, propranolol, and sotalol were 9.77, 8.13, 2.95, 1.38, 1.20, and 0.71, respectively.

Ratios of cardiac to vascular beta-receptor blockade (C:V ratios) of atenolol and propranolol were determined in pentobarbital anesthetized spontaneously hypertensive (SHR) rats. These values and the cardiovascular responses to isoproterenol were compared with those of age-matched normotensive control (Donryu, DON) rats reported previously. Mean blood pressure (BP) and heart rate (HR) were recorded. Full dose-response curves for isoproterenol were constructed in BP and HR. In vivo pD2 values of isoproterenol (potencies), were not different between DON and SHR rats, although the maximal responses to isoproterenol (efficacy) were significantly suppressed in SHR rats. C:V ratios of atenolol and propranolol in SHR rats were 7.95 and 0.44, respectively. As the values in DON rats were 9.77 and 1.20, respectively, C:V ratios of atenolol and propranolol were decreased in SHR rats. Markedly decreased C:V ratios of propranolol in SHR rats may explain its relatively weak antihypertensive action.

Antihypertensive effects of three beta-adrenergic blocking drugs, acebutolol, propranolol, and practolol were studied for 11 weeks. Spontaneously (SHR); one-clip, two-kidney (CLIP); and deoxycorticosterone and salt (DOC) hypertensive rats were used. The drugs were given orally, 100 mg/kg per day, 5 days per week before development of hypertension. Propranolol inhibited blood pressure (BP) increase significantly in SHR. Acebutolol and practolol also lowered BP in SHR. Three drugs did not affect BP in CLIP, but an apparent inhibition was seen when the results were analyzed including the cases of which BP stayed below 150 mmHg. Either of three drugs did not show antihypertensive effects in DOC. Acebutolol rather increased BP more rapidly. Practolol also increased BP slightly more rapidly. Cerebral stroke was seen in DOC. The incidences of the stroke in the groups given the solvent, acebutolol, propranolol, and practolol were 3/6, 4/7, 2/6, and 3/6, respectively. Acebutolol seemed to cause stroke earlier with the more rapid BP elevation. Acebutolol, propranolol, and practolol decreased incidence of the vascular disease in CLIP. Propranolol also decreased it in DOC. Plasma renin activity was suppressed by these drugs in SHR and CLIP. The mechanisms of antihypertensive effects of beta-adrenergic blocking drugs are unknown. The present study denies those due to inhibition of cardiac function, or renin release from the kidney. A better experimental model is necessary to study this. The possibility that acebutolol and other beta-blockers might accelerate BP elevation and incidence of stroke must be reexamined.

Plasma renin activity (PRA) was subnormal or normal in the main strain of spontaneously hypertensive rats (SHR). PRA increased greatly in the stroke-prone substrain of SHR (SHRSP) at 20-30 weeks of age. Captopril (SQ 14,225) is an orally active angiotensin-converting enzyme inhibitor. The drug acutely decreased blood pressure moderately in SHR, and markedly in SHRSP. Participation of the renin-angiotensin system in the pathogenesis of hypertension in SHR may be limited. Etiology of hypertension in connection with renal excretory function and the central and peripheral nervous system is discussed.

We report here a new type of peculiar repetitive sequence, A15T(TC)9T12, which was detected at 750 base pairs (bp) upstream of a rat calmodulin processed pseudogene by DNA sequencing of cloned DNA fragments. This sequence element could possibly form a cruciform structure with a 12-AT-pair stem, exposing (CT)9 sequences as a loop. S1 nuclease protection experiments failed to identify this element as a cruciform structure but instead detected an alternating purine pyrimidine tract at 50 bp downstream of this element. Total genomic Southern blotting showed that the rat genome contains only a few of these elements.

The structural organization of the entire rat calmodulin gene was determined by cloning and sequencing overlapping genomic and cDNA clones from rat genomic and brain cDNA libraries. The intron/exon organization was determined by direct comparison of these sequences. Rat calmodulin gene is 9000 bases long and consisted of six exons interrupted by introns of variable sizes. The first intron separates the initiation codon (ATG) from the coding region of the protein. Three out of four intron/exon junctions in the coding region reside in the middle of calcium binding subdomains and do not correlate with the quarterly divided intramolecular homology of the protein. Their positions exactly coincide with those of the corrected version of chicken calmodulin gene. The rat calmodulin gene harbors a stretch of sequences homologous to a rat middle repetitive "identifier sequence" in the middle of the third intron. Analysis of the immediate 5' upstream region detected a TATA box (TATATATAT) and three C-G boxes (CCGCCC) but not a CAT box (CCAAT). A conserved sequence (GCGCCGCGYCYYGGGGGC) was found at -125 for rat and at -204 for chicken calmodulin genes.

Effect of synthetic rat atrial natriuretic peptide (1-28) (ANP) on the cGMP content was studied using defined nephron segments of rat kidney. ANP elevates cGMP contents in glomeruli in a concentration and time-dependent manner. The increase of cGMP was observed in glomeruli, distal convoluted tubule (DCT) and cortical collecting tubule (CCT) (delta %; 279 +/- 35, 148 +/- 10 and 152 +/- 18, respectively), and no effect was observed in proximal convoluted (PCT) and straight tubule (PST). These results suggest that ANP may act directly on the tubular cells as well as glomeruli. In glomeruli, effects of ANP and carbamylcholine on cGMP contents were additive suggesting that these two agents may act on different receptors. Angiotensin II and norepinephrine failed to affect the ANP-induced cGMP production in the glomeruli.

The chemical structure of angiotensin formed by incubating the extract of the corpuscles of Stannius (CS) with homologous plasma in the Japanese goosefish , Lophius litulon , was analyzed. Goosefish CS angiotensin was proposed as [Asn1, Val5, His9 ] angiotensin I based on amino acid analysis and fluorescent peptide mapping techniques. It had the same structure as angiotensin produced by incubating the kidney extract with homologous plasma, indicating that CS angiotensin was not organ specific.

Nipradilol and prizidilol are beta-adrenoceptor blocking drugs with vasodilator action. These drugs lowered blood pressure (BP) in spontaneously hypertensive (SHR) rats acutely (24 hr) and subacutely (3 weeks) at doses of 10 and 20 mg/kg per day, p.o., respectively. Nipradilol decreased plasma renin concentration in acute and subacute studies, whereas it was unchanged with prizidilol treatment. Paradoxical effects of these drugs on BP were analyzed further: BP determined indirectly at the tail was slightly higher in SHR rats than the control, whereas BP determined directly through an aortic cannula without anesthesia, restraint, or prewarming was lower. We found that the discrepancy between BP values determined directly and indirectly was due to the increase in BP by prewarming stress during the determination by the tail cuff method.

[Asp1, Val5, Ser9]angiotensin I was synthesized by Merrifield's solid-phase procedure. The dansylated derivative of this angiotensin was cochromatographed on the TLC with the dansylated angiotensin decapeptide isolated from white leghorn fowl. Either angiotensin showed identical behavior. The per mole pressor activity of the synthetic decapeptide (in rats anesthetized with pentobarbital and treated with pentolinium) as compared to mammalian angiotensins, namely, [Ile5]angiotensin I, [Val5]angiotensin I, [Ile5]angiotensin II, and [Val5]angiotensin II, was 157, 181, 114, and 85%, respectively.

Effects of prizidilol and nipradilol (K-351), beta-adrenoceptor antagonists with vasodilator action, on blood pressure and heart rate were studied in normotensive conscious rabbits after i.v. administration. In addition, we investigated relationships between plasma drug concentrations and beta-adrenoceptor blocking activity as estimated by the inhibition of isoproterenol-induced tachycardia and vasodilator activity as assessed by the inhibition of pressor response to angiotensin II (ANG II). Prizidilol (4 mg/kg) produced a significant and sustained fall in blood pressure and a slight increase in heart rate, while hydralazine (2 mg/kg) caused the same degree of hypotension and a marked tachycardia. Nipradilol (1 mg/kg) caused a significant reduction of resting heart rate, but had no significant effect on blood pressure. Propranolol (1 mg/kg) did not affect resting blood pressure and heart rate. Hypertensive response to ANG II was significantly attenuated only by hydralazine. Isoproterenol-induced tachycardia was significantly suppressed by prizidilol, nipradilol and propranolol. Good correlations were observed between beta-adrenoceptor blocking activity and plasma drug concentrations. These data suggest that prizidilol has an advantage over hydralazine to induce less tachycardia, but still may cause a certain degree of increase in heart rate. Nipradilol has a more potent beta-adrenoceptor blocking action than propranolol, while its vasodilator action is not obvious, at least in rabbits. Plasma concentrations of prizidilol and nipradilol are good indicators for beta-adrenoceptor blocking activity, but not for vasodilator activity.

Arotinolol (S-596, ARL) is a beta-adrenoceptor blocking drug with weak alpha-adrenoceptor blocking activity, and may be classified into the fourth generation. Antihypertensive effects of ARL were studied for 12 weeks in spontaneously hypertensive (SHR) rats. Propranolol (PPL) was used as the reference drug. ARL (20 and 100 mg/kg per day, p.o.) and PPL (100 mg/kg per day, p.o.) treatments significantly decreased heart rate, within a week after the drug treatments had started and thereafter. Tail blood pressure (BP), determined by prewarming the rat at 50 degrees C for 3 min, was slightly higher in the two ARL treated groups than in the control. Tail BP was slightly lower in the PPL treated group than in the control. Mean BP determined directly at the 12th week was lower in the two ARL and PPL groups than in the control by more than 20 mmHg. Both ARL (100 mg/kg per day) and PPL (100 mg/kg per day) treatments significantly reduced incidences of the vascular lesions, and also prevented the decrease of kidney weights usually associated with mild vascular lesions. Furthermore, these treatments showed a tendency to decrease plasma renin (PRC) and aldosterone (PAC) concentrations determined 20 h after the last administration. As mean BP must be more reliable than tail BP, it was concluded that ARL (20 and 100 mg/kg per day) showed almost the same chronic antihypertensive activity in SHR rats as PPL (100 mg/kg per day). Preventive effects of ARL on development of vascular lesions also supported the above view.

Utilizing the microsurgical method of transplantation in the rat, the role of the kidney in an experimental renal hypertension due to segmental renal infarction was studied. When the infarcted kidney was transplanted into the bilaterally nephrectomized recipient, hypertension developed so long as the donor kidney was transplanted within 2 weeks after infarction. With transplantation of the normal kidney into the hypertensive rat 8 weeks after infarction, BP remained high unless the infarcted kidney was removed. PRA after transplantation, or KRA of the transplanted kidneys were not correlated to the blood pressure levels. It was suggested that the infarcted kidney has the pressor mechanism, lasting for 2 weeks or more after infarction. The infarcted kidney also has the maintenance mechanism, establishing 8 weeks after infarction.

Bometolol is a cardiospecific beta-adrenergic blocking drug. The effect on blood pressure was determined in hypertensive rats in acute and subchronic experiments. In acute experiment bometolol, 10-30 mg/kg, p.o., was given to spontaneously hypertensive rats; deoxycorticosterone and salt hypertensive rats; and two kidney, one clip hypertensive rats. Blood pressure was determined continuously for 8 h and after 24 h in unanesthetized condition. The blood pressure lowered by bometolol was dose-dependent in three types of hypertension. In subchronic experiment, bometolol at a dose of 10-30 mg/kg or 100-300 mg/kg, p.o., were given for 5 weeks to deoxycorticosterone and salt hypertensive rats; and two kidney, one clip hypertensive rats. Bometolol did not show antihypertensive effect in either type of hypertension, although bometolol decreased heart rate dose-dependently. Bometolol treatment at the above doses decreased plasma renin activity, heart and kidney weights, and incidence of vascular lesion in either hypertensive rat. Toxic symptom was seen 3 days after bometolol treatment of 300 mg/kg in deoxycorticosterone and salt hypertensive rats.

The F1 hybrids (F1) of spontaneously hypertensive rats (SHR) and Wistar rats (W), whose kidneys were transplanted from SHR at the age of 10 weeks and 20 weeks, showed significant elevation of blood pressure (BP) for 11 weeks after the transplantation. In F1 with W or F1 kidneys BP was decreased near to the normal level. F1 whose kidneys were transplanted from SHR or W showed low renin activity both in plasma and the kidney. It is suggested that BP of SHR is probably determined by the renal pro-hypertensive factor(s) other than renin influencing on sympathetic nerves through central nervous systems.

A prospective survey on the comparative studies of the renin-angiotensin system (RAS) was made at the final session of the Japan-U.S. Cooperative Seminar, held on November 14--19, 1977, at Jichi Medical School, Tochigi-ken, Japan. Strategies and tactics of the research have been clarified. In morphological studies, the strategy is to clarify structure of the juxtaglomerular apparatus in representative species of vetebrate classes. In chemical studies, each component of the RAS must be established in each vertebrate class. Since we now know the existence of severel new angiotensins in vertebrates, their amino-acid sequences must be determined. They must be synthesized in sufficient purity. It is necessary to establish the international purity and assay criteria for synthetic angiotensins. In comparative studies on physiological aspects, the strategy is to establish the physiological roles of the RAS in vertebrate classes. Controls of blood pressure, of aldostrone secretion, of glomerular filtration rate and tubular transport, and of drinking and intestinal absorpiton are the candidates. Physiological roles of extrarenal renin as well as renal one are interesting. A renin-like substance in the corpuscles of Stannius in the teleosts has been identified preliminarily as hypocalcin. Many frontiers exist in these fields, and one can expect fruitful contributions to research of the cardiovascular disease in humans through comparative studies of the RAS.

As a discrepancy exists between two reports from our laboratory on plasma renin activity (PRA) in spontaneously hypertensive rats (SHR), we undertook to examine the reasons for this discrepancy. PRA in SHR at 15 weeks of age was determined by two different methods, utilizing the procedures of Boucher et al or of Haber et al. For normotensive controls, Donryu (DON) and Wistar-Kyoto (WKY) rats were used. By Boucher's method, PRA in SHR was significantly lower than in DON, but did not differ from WKY. By Haber's method, PRA in SHR did not significantly differ from DON or WKY. However, the value in DON was significantly higher than in WKY. The present study basically confirmed our previous results, concluding that PRA in SHR at 15 weeks of age is within the normal range. A previous suggestion that PRA in SHR is suppressed as a result of blood pressure elevation must be revised, because it was derived from results considering only the DON strain as the control.

Plasma and kidney renin activity (PRA, KRA) were determined in the spontaneously hypertensive (SHR) rats, the stroke-resistant and -prone substrains (SHRSR, SHRSP) from 5 to 30 weeks of age. Results were compared with those of two normotensive strains, Wistar-Kyoto (WKY) and Donryu (DON) rats. PRA in the SHRSP at 20 and 30 weeks of age were significantly increased when compared to other strains of rats (P < 0.01). In SHRSP rats at these ages, blood pressure exceeded the critical level of 220 mmHg and cerebral lesions were observed in 41% at autopsy. There were no significant differences in PRA among other hypertensive and normotensive strains. KRA in three substrains of the SHR were normal or subnormal as compared to WKY and DON rats. These results indicate that a direct role of the renin-angiotensin system in the SHR and its substrains can be excluded in the initiation and the maintenance of hypertension. However, the activated renin-angiotensin system in SHRSP rats in the course of malignant hypertension at 20 weeks of age and later, could participate in raising blood pressure above the levels of the SHR and SHRSR. Considering out data and others, there are many similarities in renin profile between the SHR and its two substrains, and human essential hypertension in which PRA can be classified as low, normal or high.