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 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.

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.

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.

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.

Synthesis and specific pressor activity of [1-aspartic acid,5-valine,9-serine]angiotensin I ("fowl angiotensin I").

[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.

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.

Blood pressure regulation by angiotensin in the spontaneously hypertensive rats.

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.

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.

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.

Renin-angiotensin system in spontaneously hypertensive rats.

Plasma and kidney renin activities (PRA, KRA) were determined in male spontaneously hypertensive rats (SHR), the inbred strain of F27-30. Blood samples of 0.5 ml for PRA determination were obtained through a cannula inserted into the abdominal aorta without anesthesia to minimize renin release from the kidney. The PRA was 30 wk. The KRA of SHR at age 10, 20, and 30 wk was also lower, confirming a previous report. At 5 wk of age, KRA was slightly higher than that of normal controls. At 50 wk of age, PRA and KRA were significantly lower in SHR (F25) from a random-bred colony than in normal Donryu rats. It is suggested that the renin-angiotensin system is suppressed in SHR as a compensatory reaction against blood pressure elevation.

The ratio of cardiac to vascular beta-receptor blockade of atenolol and propranolol in spontaneously hypertensive rats in vivo.

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.

Variation of plasma and kidney renin activities among substrains of spontaneously hypertensive rats.

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.

Sympathetic nervous activity in renal and DOC hypertensive rats.

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.