Structural organization of calmodulin genes in the rat genome.

In summary, we present a list of phage clones we have obtained from rat genomic libraries (from lamba SC1 to lambda SC31, lambda WC1 and lambda WC40) together with cDNA clones we have obtained from a rat brain cDNA library (PRCM1,5,3 and 4). pRCM5 corresponds to 4.0 kb mRNA species observed primarily in skeletal muscle. These clones can be classified into three groups. They belong to three bona fide calmodulin genes with five to six exons called CaM I, CaM II and CaM III and four intronless retropseudogenes, one derived from CaM I and three derived from CaM II. We have not obtained retropseudogenes for CaM III so far. These three bona fide genes are transcribed into multiple sized mRNA species in a tissue-specific manner, that is, CaM I is ubiquitous, CaM II is transcribed mainly in brain and CaM III is transcribed primarily in brain and skeletal muscle. Four retropseudogenes do not appear to be transcribed. They are probably relics of inactivated genes. The physiological meanings of multiple calmodulin mRNA species and mechanisms of transcriptional regulation of these three bona fide genes will be the main subjects of our future experiments.

Genes and pseudogenes for calmodulin in the spontaneously hypertensive rat.

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.

Antihypertensive treatment in spontaneously hypertensive rats with streptozotocin-induced diabetes mellitus.

Recent clinical reports have suggested that hypertension accelerates the progress of diabetic nephropathy and retinopathy, whereas antihypertensive treatments may retard them. Thus, the effect of antihypertensive treatment in diabetes mellitus with hypertension was evaluated in rats. A model of diabetes mellitus with hypertension has been developed in spontaneously hypertensive (SHR) rats by unilateral nephrectomy and streptozotocin (STZ, 30 mg/kg, i.v. treatment). The rats were treated with four antihypertensive drugs orally for 12 weeks thereafter. STZ treatment induced chronic hypeglycaemia (300-400 mg/dl), decreased body weight and heart rate, and caused vascular changes of ophthalmic fundi and cataracta. The kidney of these rats showed proliferative changes such as periarteritis nodosa, hyperplasia, or fibronecrosis of the arterioles, exudative changes, mesangial proliferation, or thickening of the basement membrane of the glomeruli. Enalapril (10 mg/kg per day) and remipril (Hoe 498) (1 mg/kg per day), converting enzyme inhibitors, or arotinolol (20 mg/kg per day), a beta-adrenoceptor blocking drug, decreased blood pressure, prevented the development of renal and ocular lesions, and tended to increase creatinine clearance. Nisoldipine (3 mg/kg per day), a calcium-entry blocking drug, tended to decrease blood glucose, and prevented the decrease of body weight and development of ocular lesions. In conclusion, antihypertensive treatments were effective in preventing the progress of diabetic retinopathy and nephropathy, and renal insufficiency in this animal model.

Does so-called streptozocin hypertension exist in rats?

Although the existence of so-called streptozocin hypertension seems well established, some reports have indicated that no rise in blood pressure (BP) occurred after streptozocin treatments. To ascertain the streptozocin-induced BP response, normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were treated with streptozocin, 40 to 45 and 35 mg/kg i.v., respectively, and BP was determined directly and indirectly every week for 3 to 4 weeks. Direct mean BP was determined without anesthesia or restraint through a cannula inserted into the rat's abdominal aorta. Indirect BP was determined at the tail without anesthesia after prewarming the rat in a holder. Compared with control values, indirect BP increased significantly in diabetic WKY 2 weeks after streptozocin treatment. In contrast, direct BP of these rats decreased, compared with control values. Indirect BP of diabetic SHR was as high as that of the controls, whereas direct BP of diabetic SHR decreased significantly 1 week after the treatment and thereafter, compared with control values. These discrepancies between the direct and indirect BP values may be caused by severe emaciation of diabetic rats. Extra pressure in the cuff may be necessary to occlude the bloodstream. These results indicate that under these conditions the value of BP obtained by the direct measurement is more reliable than that by the indirect one; therefore, we concluded that so-called streptozocin hypertension does not exist.

Multiple calmodulin mRNA species are derived from two distinct genes.

We have observed three calmodulin mRNA species in rat tissues. In order to know from how many expressed genes they are derived, we have investigated the genomic organization of calmodulin genes in the rat genome. From a rat brain cDNA library, we obtained two kinds of cDNAs (pRCM1 and pRCM3) encoding authentic calmodulin. DNA sequence analysis of these cDNA clones revealed substitutions of nucleotides at 73 positions of 450 nucleotides in the coding region, although the amino acid sequences of these calmodulins are exactly the same. DNA sequences in the 5' and 3' noncoding regions are quite different between these two cDNAs. From these results, we conclude that they are derived from two distinct bona fide calmodulin genes, CaMI (pRCM1) and CaMII (pRCM3). Total genomic Southern hybridization suggested four distinct calmodulin-related genes in the rat genome. By cloning and sequencing the calmodulin-related genes from rat genomic libraries, we demonstrated that the other two genes are processed pseudogenes generated from the CaMI (lambda SC9) and CaMII (lambda SC8) genes, respectively, through an mRNA-mediated process of insertions. Northern blotting showed that the CaMI gene is transcribed in liver, muscle, and brain in similar amounts, whereas the CaMII gene is transcribed mainly in brain. S1 nuclease mapping indicated that the CaMI gene produced two mRNA species (1.7 and 4 kilobases), whereas the CaMII gene expressed a single mRNA species (1.4 kilobases).

Structure of a gene for rat calmodulin.

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.

Altered vitamin D metabolism in the spontaneously hypertensive rat.

To test the possible impairment of vitamin D metabolism in hypertension, we studied the effect of parathyroid hormone (PTH) on the renal production of 1,25-dihydroxyvitamin D [1,25(OH)2D] in spontaneously hypertensive rats (SHR) before (at 4 weeks of age) and after (at 12 weeks of age) the onset of hypertension. Basal serum of 1,25(OH)2D was normal in SHR at both ages. At 4 weeks of age, rise in serum 1,25(OH)2D following PTH injection (50 U subcutaneously every 2 h, four times) was also normal in SHR. By contrast, at 12 weeks of age it was approximately one-third of that in Wistar-Kyoto rats (WKY) in parallel with an attenuated response to PTH of renal production of 1,25(OH)2D. Basal 1,25(OH)2D production by the kidney in SHR was higher than that in WKY at both ages, which was abolished by thyroparathyroidectomy but not by parathyroidectomy. These data demonstrate that altered vitamin D metabolism exists even before the onset of hypertension in SHR.

A peculiar repetitive sequence in the rat genome.

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.

Structure of rat calmodulin processed genes with implications for a mRNA-mediated process of insertion.

Two distinct processed calmodulin genes of rat (lambda SC8 and lambda SC9) were identified, cloned and their DNA sequences determined. The existence of direct repeats of 19 base-pairs for lambda SC8 or 9 base-pairs for lambda SC9 at both ends of the coding plus non-coding regions suggested a possible involvement of a mRNA-mediated process of insertion. Total genomic Southern hybridization suggested the existence of at least three different calmodulin-related genes in the rat genome. The other gene was the bona fide calmodulin gene (lambda SC4) which was split into at least five exons. lambda SC9 contained insertions of one nucleotide and two 17 base-pair direct repeats in the coding region. These insertions cause frameshift mutations probably preventing it from encoding a functional calmodulin. It also carried an insertion of a rat middle repetitive sequence, identifier sequence (IDS: Sutcliffe et al., 1982) in the 3'-non-coding region. Otherwise, it consisted of an almost identical DNA sequence to that of the bona fide calmodulin gene (lambda SC4), including the 3'-non-coding region down to the poly(A) recognition signal, A-A-T-A-A-A. On the other hand, lambda SC8 did not possess frameshift mutations in the coding region, and hence was capable of encoding a functional protein. In fact, a probe specific to the lambda SC8 sequence identified a band in Northern blotting whose size was 300 nucleotides smaller than that of authentic calmodulin mRNA. Comparison of the nucleotide sequences showed that only the coding regions of these two processed genes were homologous, indicating that the divergence of these two processed genes from the common ancestor calmodulin was an ancient event.

Atrial natriuretic peptide elevates cGMP contents in glomeruli and in distal tubules of rat kidney.

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.

Effects of enalapril and captopril on urinary excretion of kinins and electrolytes in stroke-prone spontaneously hypertensive rats.

Urinary excretion of kinins in stroke-prone spontaneously hypertensive (SHRSP) rats was unchanged during oral enalapril (10 mg/kg) or captopril (30 mg/kg) treatment once a day for 8 days compared to vehicle treatment. However, a significant decrease in urinary kinin excretion was observed on the 5th and 7th day compared to the pretreatment value in rats treated with enalapril. Both enalapril and captopril produced a significant reduction in blood pressure when compared to the vehicle. These findings provide no positive evidence to support the hypothesis of possible involvement of renal kinins in the antihypertensive effect of converting enzyme inhibitors in SHRSP rats.

Chronic effects of enalapril on blood pressure, stroke, plasma renin, urinary electrolytes and PGE2 excretion in stroke-prone spontaneously hypertensive rats.

Antihypertensive effect of enalapril (MK-421), an orally active non-sulfhydryl-containing converting enzyme inhibitor, was examined in stroke-prone spontaneously hypertensive (SHRSP) rats. The treatment was started at 14-15 weeks of age with tail blood pressure over 240 mmHg and was continued for 11 weeks. We used captopril as the reference drug. The dose of enalapril and captopril was 10 and 30 mg/kg per day, p.o., respectively. Enalapril showed a sustained antihypertensive effect from the 1st to the 11th week of the treatment. This antihypertensive effect was substantiated by the good increase in body weight; decrease in heart weight; decrease in incidences of vascular disease, nephrosclerosis, stroke and death. Enalapril treatment also prevented the increases in urine volume, and excretion of osmotically active solutes, Na, Cl and K with age. Captopril treatment showed about the same antihypertensive effect. No side effects were seen in the enalapril or captopril treated group. The antihypertensive potency of enalapril was about 3 times more than that of captopril. Enalapril and captopril slightly increased plasma renin concentration. Urinary excretion of PGE2 was not changed by enalapril or captopril treatment. These results clearly demonstrate the efficacy of long-term treatment with enalapril to prevent development of malignant hypertensive cardiovascular disease in SHRSP rats.

An analysis of blood pressure effects of nipradilol and prizidilol in normotensive and spontaneously hypertensive rats.

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.

Chronic effects of a beta-adrenoceptor blocking drug, 4-[3-(tert-butylamino)-2-hydroxypropoxy]-N-methylisocarbostyril hydrochloride (N-696), in hypertensive rats.

4-[3-(tert-Butylamino)-2-hydroxypropoxy]-N-methylisocarbostyril hydrochloride (N-696) is a new beta-adrenoceptor blocking drug with direct vasodilatory activity, and may be classified into the fourth generation. Antihypertensive effects of N-696 were studied for 12 weeks in spontaneously (SHR), two kidney, one clip (CLIP), and deoxycorticosterone-salt (DOC) hypertensive rats. Propranolol (PPL) was used as the reference drug. In indirect blood pressure (BP) determination at the tail, milder prewarming condition was employed to observe antihypertensive effects clearly. Rats were prewarmed in rat holders on a hot plate for 30-60 min. Surface temperature of the hot plate was 35-45 degrees C. N-696 (20 mg/kg per day p.o.) and PPL (100 mg/kg per day, p.o.) treatments significantly decreased heart rate (HR) and maximum BP determined indirectly in SHR rats, even at the early stage of the experiments. These antihypertensive effects were shown also by mean BP determined directly at the 12th week. N-696 and PPL treatments showed no significant antihypertensive effects in CLIP rats, and slight antihypertensive effects in DOC rats. N-696 treatments showed a tendency to decrease plasma renin concentration (PRC) in SHR and DOC rats, whereas PPL treatments significantly decreased PRC in these hypertensive rats. N-696 and PPL treatments prevented nephrosclerosis and vascular lesions in SHR and DOC rats only slightly.

Chronic effects of arotinolol (S-596) in spontaneously hypertensive rats.

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.

Pharmacodynamic and pharmacokinetic studies on prizidilol and nipradilol (K-351), antihypertensive drugs with combined vasodilator and beta-adrenoceptor blocking actions, in rabbits.

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.

Chemical structure of angiotensin formed by incubating plasma with the corpuscles of Stannius in the Japanese goosefish, Lophius litulon.

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.

Chemical structure of angiotensin in the turtle, Pseudemys scripta.

The chemical structure of angiotensin generated by incubating kidney extract with homologous plasma from the turtle, Pseudemys scripta, has been analyzed. The turtle angiotensin was proposed to be [Asp1, Val5, His9] ANG I by its amino acid composition and by its fluorescent peptide mapping. It was the same structure as angiotensin I in the ox and the sheep. The N-terminal amino acid of the turtle angiotensin was not blocked, unlike that of another reptilian angiotensin found in the snake, Elaphe climacophora.