Cardiac beta myosin heavy chain diversity in normal and chronically hypertensive baboons.

We have identified two distinct beta-myosin heavy chains (MHCs) present in baboon myocardium by electrophoresis in gradient pore gels and by Western blots with anti-MHC MAb. The two beta-MHCs have molecular masses of 210 and 200 kD and share several antigenic determinants including an epitope recognized by a beta-MHC-specific MAb. A fivefold increase in the level of the 200-kD beta-MHC was observed in the hypertrophied left ventricles of baboons with chronic (5.3 +/- 0.7 yr) renal hypertension. A 60% increase (P less than 0.01) in BP and a 100% increase (P less than 0.001) in left ventricular mass to body weight ratio occurred in hypertensive baboons compared with normotensive animals. The Ca2+-activated myosin ATPase activity in hypertrophied left ventricles was decreased by 35% (P less than 0.05) compared with controls. Normal levels of the 200-kD MHC were detected in the right ventricles and intraventricular septa of the hypertensive animals. These data suggest that cardiac MHCs of primates may exist in alternative molecular forms that are indistinguishable by nondenaturing gel electrophoresis and that increased concentration of a second beta-MHC is associated with ventricular hypertrophy (r = 0.55). The functional significance and mechanisms that control the concentration of beta-MHC subspecies remain to be determined.

Metabolic clearance of immunoreactive vasopressin and immunoreactive [131I]iodo vasopressin in the hypophysectomized dog.

Fourteen acutely hypophysectomized, anesthetized dogs were given a constant infusion of arginine vasopressin (AVP) and 131I-labeled arginine vasopressin ([131I]AVP). After 90 min, 3 blood samples were collected at 15 min intervals for determination of total body clearances of immunoreactive AVP and immunoreactive [131I]AVP. Seven dogs were then nephrectomized. Ninety minutes later, a second set of 3 blood samples was collected at 15 min intervals for clearance measurements in these and the 7 time-control dogs. Prenephrectomy AVP clearance averaged 5.1+/-1.0 ml/min-kg (mean +/- SE, n=7), and the 210-240 min postnephrectomy AVP clearance average 4.9+/-0.8. The 90-120 min average clearance in the time-control dogs was 6.1+/-0.9 ml/min-kg (n=7) and AVP clearance in these dogs increased (P less than 0.01) with time to 7.3+/-0.9 ml/min-kg during the 210-240 min period of constant infusion. Although the postnephrectomy AVP clearance was not significantly changed from prenephrectomy levels, it was significantly lower (P less than 0.05) than the 210-240 min average clearance in the time-controls. Clearance of [131I]AVP was 3.3+/-0.2 ml/min-kg (n=7) before nephrectomy and 2.9+/-0.2 ml/min-kg after nephrectomy. This was a significant 12% reduction (P less than 0.01). [131I]AVP clearance in the time control dogs was 3.9+/-0.3 during 90-120 min of infusion and 3.9+/-0.4 during 210-240 min of infusion. [131I]AVP clearance before nephrectomy was 79+/-12% of AVP clearance (P less than 0.005) and afther nephrectomy was 74+/-16% of AVP clearance (P less than 0.05). Although these results might suggest that [131I]AVP clearance is at least a qualitative indicator of AVP clearance, there was no significant correlation (P less than 0.20) between AVP clearance and [131I]AVP clearance.

Volume control of plasma antidiuretic hormone concentration following acute blood volume expansion in the anesthetized dog.

Acute volume expansion was produced in 9 dogs by infusing a lactated Ringer's solution at 1 ml/kg/min in a volume estimated to increase blood volume by 20%. Volume expansion was maintained by replacing urinary fluid losses with equal volumes of the Ringer's solution. Following volume expansion, the effects of a slow, nonhypotensive hemorrhage on plasma antidiuretic hormone concentration (PADH) were determined and compared to a group of 9 normovolemic dogs subjected to the same hemorrhage procedure, in order to determine if volume receptor control of ADH release would adapt to acute increases in blood volume. Ringer's infusion significantly increased blood volume to 95.2 +/- 3.1 ml/kg (mean +/- SE; P less than 0.01) when compared to a mean normovolemic blood volume of 77.6 +/- 3.4 ml/kg. Volume expansion was associated with a significantly lower PADH (3.2 +/- 1.6 muU/ml) than that in normovolemic dogs (5.7 +/- 1.2 muU/ml; p less than 0.05). Significant increases in PADH (P less than 0.05) occurred in both groups of dogs after 20 and 40 minutes of a continuous, nonhypotensive hemorrhage (0.40 to 0.45 mg/kg/min. Hemmorrhage was also associated with significant decrease in effective left atrial pressure in both groups of dogs after 5, 10, 20, and 40 minutes of hemorrhage (P less than 0.01). There were no significant differences between the two groups of dogs nor were there any significant changes during the experiment within each group for mean arterial blood pressure, arterial pulse pressure, plasma osmolality, plasma sodium concentration and plasma potassium concentration. Effective left atrial pressure and PADH were found to be exponentially correlated with blood volume in bothy hypervolemic and normovolemic dogs. Analysis of covariance of these correlations suggested that the hypervolemic dogs exhibited the same exponential changes in PADH and effective left atrial pressure with decreased blood volume as in the normovolemic dogs. It is concluded that acute volume expansion does not alter volume control of plasma ADH concentration.

Suppression of renin and aldosterone by small amounts of DOCA in normal man.

Previous investigators have suggested that low renin hypertension may be due to an unknown mineralocorticoid. This investigation was designed to simulate the effect of an unknown mineralocorticoid by administration of small amounts of desoxycorticosterone acetate (DOCA) in three normal subjects. The response of 2-h upright plasma renin activity (PRA), plasma aldosterone concentration (PA), and urinary aldosterone excretion (UA), as well as extracellular fluid volume, (ECFV) was determined on a high and low salt diet before and during the administration of DOCA for 13 days. After 9 days of DOCA, ECFV increased approximately 2 liters and PRA decreased to levels found in our patients with LRH. PA and UA decreased appropriately as PRA was suppressed. We would expect an unknown mineralocorticoid to have similar effects on the reninangiotension-aldosterone system. Thus, these results would suggest that in LRH the normal levels of PA and UA are inappropriately elevated in relation to the low PRA.

Pressor responsiveness to vasopressin in the rat with DOC-salt hypertension.

To further characterize the role of vasopressin in DOC-salt hypertension, four groups of unilaterally nephrectomized rats were studied: control rats given no further treatment, rats treated with DOC and given 1% saline to drink, or rats treated with only DIC or 1% saline had similar pressor responses to exogenous vasopressin and angiotensin II. Within the DOC-salt group, two populations of rats were identified: one with normal pressor responsiveness to vasopressin, and one with markedly enhanced pressor responsiveness to vasopressin. Incidence of enhanced responsiveness increased with duration of treatment. Urinary excretion of vasopressin was elevated in the 1% saline and DOC-salt groups after 1 week of treatment, and in the DOC group after 4 weeks. However, the plasma vasopressin concentration was elevated only in the rats treated with both DOC and saline. It is suggested that vasopressin is essential for the expansion of blood volume in the early stages of DOC-salt hypertension, and functions as a direct pressor agent only in the later stages.

Selective breeding to develop lines of baboons with high and low blood pressure.

Lines of baboons with high and low blood pressure were developed by selective breeding. Blood pressure was measured in 456 adult feral baboons under ketamine immobilization by direct arterial cannulation. Males with blood pressures two standard deviations and females with blood pressures one standard deviation above and below the cumulative mean were selected as progenitors. High males were mated with high females and low males were mated with low females. We measured blood pressure and plasma renin activity on 100 progeny, 54 males and 46 females, greater than 44 months of age with an abbreviated tether protocol and software program for data collection. Mean systolic and diastolic nighttime pressures for the high line were 126/72 and for the low line were 114/65 mm Hg. Line differences for systolic (12 mm Hg) and for diastolic (7 mm Hg) pressures were significant (p < 0.001). The line difference for plasma renin activity (1.1 [ng/mL]/hr) was not significant. Progeny pressures ranged from 84/49 to 191/126 mm Hg. There was no sex effect on blood pressure or plasma renin activity line differences. Heritability of systolic pressure was 0.46 +/- 0.19 and of diastolic pressure was 0.32 +/- 0.19. These results indicate that, by selective breeding and rigorous measurement of blood pressure, lines of baboons with significant difference in blood pressure can be developed.

Linkage of essential hypertension to the angiotensinogen locus in Mexican Americans.

Essential hypertension has been linked to a highly polymorphic marker at the angiotensinogen locus, and association with a polymorphism in this locus has been found in some populations. We tested the hypothesis that these same polymorphic markers are linked to essential hypertension in Mexican Americans. The data comprised all the affected relative pairs in 46 extended families chosen at random from a low-income barrio in San Antonio. Specifically, we searched for linkage by testing for excessive marker alleles shared identical by descent (IBD) among hypertensive relative pairs. When women taking oral contraceptives or hormones were excluded, the affected relative pairs shared a significant excess of alleles IBD for the highly heterozygous GT repeat polymorphism (P=.038) and were marginally significant for the M235T variant (P=.079), which has a much lower heterozygosity (0.43 versus 0.85 for the GT repeat). We also assayed plasma levels of angiotensinogen and, using likelihood methods, found no significant association (P=.43) between plasma levels of angiotensinogen and M235T genotypes. These results support the linkage of essential hypertension to the angiotensinogen locus but do not indicate a specific role for the M235T variant.

The importance of vasopressin in the development and maintenance of DOC-salt hypertension in the rat.

Experiments were performed to determine the role of vasopressin in deoxycorticosterone (DOC)-salt hypertension. In order to determine if vasopressin is necessary for the development of DOC-salt hypertension, rats with hereditary diabetes insipidus (DI) and normal Long-Evans rats (LE) were unilaterally nephrectomized, treated with DOC Pivalate (30 mg/kg . week) and given saline to drink for 8 weeks. A second group of DI rats were unilaterally nephrectomized, but received no treatment. Systolic blood pressure (SBP) increased 40 mm Hg in the LE group (p less than 0.01) but failed to increase significantly in either DI group. Urinary excretion of vasopressin (UADHV) and SBP were measured in unilaterally nephrectomized LE rats treated with DOC and salt (DOC-LE), salt alone (NaCl-LE) and untreated rats (H2O-LE). The UADHV was elevated in DOC-LE (p less than 0.01) and NaCl-LE (p less than 0.05), but only the DOC-LE rats became hypertensive. Finally, the I.V. injection of analogs of vasopressin, which block its pressor but not antidiuretic activity, lowered mean arterial blood pressure 27 +/- 5 mm Hg in 11 conscious DOC-salt hypertensive rats. It is concluded that vasopressin plays a major role as a pressor agent in both the onset and maintenance of DOC-salt hypertension.

A plant defensive cystatin (soyacystatin) targets cathepsin L-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera).

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.

Plasma vasopressin concentration in high sodium renal hypertension.

One-kidney, figure-8 renal wrapped and sham-operated rats maintained on high sodium intake were studied to determine plasma concentrations of vasopressin during the onset of hypertension. Animals were chronically prepared with femoral artery and vein catheters. Arterial blood samples were taken from conscious rats before and 3 days after renal wrap or sham operation while donor blood was simultaneously infused intravenously. Three days after surgery, arterial pressure, plasma osmolality and plasma vasopressin concentration increased significantly in the renal wrapped animals and remained unchanged in the sham-operated rats. Ganglionic blockade with hexamethonium and atropine produced equivalent decreases in arterial pressure and increases in plasma vasopressin concentration in the two groups of rats. Subsequent administration of the V1 vasopressin antagonist, d(CH2)5Tyr(Me)AVP, caused a significantly greater fall in arterial pressure in the hypertensive rats. These results provide further evidence for a contribution of vasopressin to sodium-dependent hypertension.

Vasopressin release during ventriculo-cisternal perfusion with prostaglandin E2 in the dog.

In an attempt to determine whether prostaglandin E2 (PGE2) can act centrally to affect the release of vasopressin (ADH), the ventriculo-cisternal system of anaesthetized dogs was perfused with PGE2. When PGE2 was perfused at a rate of 76-4 ng/min (0-19 ml/min), the plasma ADH concentration was unchanged. However, perfusion of PGE2 at a rate of 152-8 ng/min (0-19 ml/min) resulted in a significant increase in the plasma ADH concentration from the control value of 9-0 +/- 2-2 (S.E.M.) to 18-8 +/- 3-9 muu./ml at 10 min and to 41-0 +/- 16-7 muu./ml at 30 min after the start of the perfusion. There were no changes in arterial blood pressure, rectal temperature, plasma osmolality, and the plasma concentrations of sodium and potassium. In additional experiments, i.v. injection of indomethacin (2 or 20 mg/kg) decreased the plasma ADH concentration by approximately 50%. Although this finding is consistent with a role of PGE2 in the control of ADH release, it could also have been due to the observed increases in arterial blood pressure and effective left atrial pressure. Plasma renin activity was unchanged in the indomethacin experiments. It is concluded that PGE2 can act in the central nervous system to stimulate ADH release.

Aortic baroreceptor deafferentation in the baboon.

Previous animal studies have indicated that removal of the aortic baroreceptors causes a moderate increase in arterial pressure that is not fully buffered by receptors in the carotid sinus. However, the role of these separate baroreceptors in the conscious nonhuman primate has not been examined. To address this question, adult male baboons were chronically maintained on a tether system that permitted them to move freely about their cage. With this system, arterial pressure and heart rate could be monitored continuously over 24-h periods with periodic drug administration to test cardiovascular function. Control values of arterial pressure and heart rate were 85.6 +/- 4.0 mmHg and 77.5 +/- 2.9 beats/min, respectively. Following removal of the aortic baroreceptors, arterial pressure rose to 104.6 +/- 5.5 mmHg and heart rate increased to 117.9 +/- 3.1 beats/min. The variability of these parameters did not change following denervation. There was, however, a suppression of the arterial pressure-heart period relationship and an augmentation in the depressor response to ganglionic blockade with hexamethonium. These data indicate that removal of the aortic baroreceptors causes a reduction in the sensitivity of the heart rate baroreflex and subsequent increase in arterial pressure that is a result of an increased sympathetic nervous system function.

Evidence of an endogenous forebrain GABAergic system capable of inhibiting baroreceptor-mediated vasopressin release.

In conscious rats, intracerebroventricular (i.c.v.) injections of gamma-aminobutyric acid (GABA), a GABA-uptake inhibitor (nipecotic acid), and artificial CSF (aCSF) were restricted to forebrain regions and their effect on baroreceptor-mediated arginine-vasopressin (AVP) release was studied. AVP release was stimulated by the hypotension resulting from combined treatment with a converting enzyme inhibitor (CEI) and chlorisondamine (CHLOR), a ganglionic blocking agent. CEI + CHLOR reduced mean arterial pressure (MAP) from 118 +/- 2 to 63 +/- 2 mm Hg, but pressure then rose to a compensated level of 78 +/- 1 mm Hg. The compensation in MAP was shown to be AVP-dependent at the end of the experiment since the vascular AVP antagonist, d(CH2)5Tyr(Me)AVP, reduced MAP from 78 +/- 1 to 63 +/- 1 mm Hg. While AVP was contributing to MAP maintenance, GABA (15, 50 and 150 micrograms) caused dose-related reductions in MAP (5 +/- 1.7 +/- 1 and 11 +/- 2 mm Hg, respectively). Nipecotic acid (3-350 micrograms) also caused dose-related reductions in MAP (from 3 +/- 1 to 15 +/- 2 mm Hg), while aCSF had no effect on MAP. Pretreatment with d(CH2)5Tyr(Me)AVP, antagonized completely the depressor effects of GABA and nipecotic acid. In other rats, blood samples were taken to measure the changes in plasma AVP concentrations (pAVP) induced by CEI + CHLOR and subsequent treatment with aCSF or nipecotic acid (175 micrograms). Hypotension induced by CEI + CHLOR caused a significant increase in pAVP. Forebrain-restricted nipecotic acid significantly suppressed pAVP (61 +/- 8% reduction; P less than 0.05 vs aCSF). These data provide evidence of an endogenous forebrain GABAergic system which, when activated, can inhibit baroreceptor-mediated AVP release.

Noninvasive measurement of pulsatile blood volume changes. Its usefulness in peripheral vascular disease.

A clinical trial was made of a new noninvasive technic for the measurement of arterial blood volume changes in a limb segment. The instrument employed is small, portable, provides a digital display in cc/min, is readily calibrated, gives reproducible values of the segmental phen be used to screen patients with peripheral vascular disease and to assess postoperative results.

Effect of 30 per cent maternal nutrient restriction from 0.16 to 0.5 gestation on fetal baboon kidney gene expression.

Previous studies in rodents and sheep show that maternal nutrient restriction during pregnancy alters fetal renal development. To date, no studies using fetal baboon RNA with human Affymetrix gene chips have been published. In the present study we have (1) evaluated the specificity of the Affymetrix human gene array 'Laboratory on a Chip' system for use with fetal baboon mRNA and (2) investigated the effects of moderate maternal global nutrient restriction (NR; 70% of ad libitum animals) from early (30 days gestation (dG)) to mid-gestation (90 dG; term = 184 dG) on the fetal baboon kidney. Morphometric and blood measurements were made on 12 non-pregnant baboons before they were bred. All baboons were fed ad libitum until 30 days pregnant, at which time six control baboons continued to feed ad libitum (control - C) while six received 70% of the C diet on a weight adjusted basis. Fetal kidneys were collected following caesarean section at 90 dG, with samples flash frozen and fixed for histological assessment. Fetal hip circumference was decreased in the NR group (68 +/- 2 versus 75 +/- 2 mm), while fetal body weight and all other measurements of fetal size were not different between C and NR at 90 dG. Maternal body weight was decreased in the NR group (12.16 +/- 0.34 versus 13.73 +/- 0.55 kg). Having established the specificity of the Affymetrix system for fetal baboon mRNA, gene expression profiling of fetal kidneys in the context of our maternal nutrient restriction protocol shows that NR resulted in a down-regulation of genes in pathways related to RNA, DNA and protein biosynthesis, metabolism and catabolism. In contrast, genes in cell signal transduction, communication and transport pathways were up-regulated in the NR group. These changes indicate that even a moderate level of maternal global NR impacts fetal renal gene pathways. Our histological assessment of renal structure indicates decreased tubule density within the cortex of NR kidneys compared with controls. The number of glomerular cross-sections per unit area were unaffected by NR, suggesting that tubule tortuosity and/or tubule length was decreased in the NR kidney. Taken together the changes indicate that NR results in accelerated fetal renal differentiation. The negative impact of poor maternal nutrition on the fetal kidney may therefore be in part due to shortening of critical phases of renal growth resulting in decreased functional capacity in later life. These findings may have important implications for postnatal renal function, thereby contributing to the observed increased predisposition to hypertension and renal disease in the offspring of nutrient restricted mothers.

Effectiveness of recombinant soybean cysteine proteinase inhibitors against selected crop pests.

Three recombinant soybean cysteine proteinase inhibitors (rSCPIs), L1, R1 and N2, were assessed for their potential to inhibit the growth and development of three major agricultural crop pests known to utilize digestive cysteine proteinases: Western corn rootworm (Diabrotica virgifera virgifera, WCR), Colorado potato beetle (Leptinotarsa decemlineata, CPB) and cowpea weevil (Callosobruchus maculatus, CW). In vitro experiments showed that cysteine proteinase activities in the crude gut extracts of the WCR, CPB, and CW were inhibited to various degrees by the three rSCPIs. Of the three rSCPIs tested, N2 was most effective in inhibiting the crude gut extract of WCR, CPB, and CW (50% inhibition at 5 x 10(-8), 5 x 10(-8), and 3 x 10(-7) M, respectively). The L1 was the least potent of the three CPIs tested, with 50% inhibition at 5 x 10(-6) M of the crude gut extracts of WCR. Results of in vivo experiments conducted to assess the effect of the three rSCPIs on the vital growth parameters of WCR, CPB and CW were consistent with results of the in vitro experiments.

"Normal" blood pressure in chimpanzees.

During routine physical examination under ketamine sedation, 140 blood pressure measurements from male and 170 from female chimpanzees were derived over a period of 3 yr. Statistical analysis revealed generally no differences between the sexes but significant increases of both systolic and diastolic blood pressure with increasing age. The data obtained may constitute a useful management tool with which to identify hypertensive chimpanzees and suggests that this animal species could be a model for hypertension research.

Renal vasopressin clearance with reductions in renal blood flow in the dog.

These! experiments were designed to evaluate directly the effects of a reduction in renal blood flow on the renal metabolism of vasopressin (ADH) in the anesthetized dog...

Vasopressin release during nonhypotensive hemorrhage and angiotensin II infusion.

These experiments were designed to determine whether angiotensin II (AII) could potentiate the increase in plasma vasopressin (ADH) concentration produced by continuous, nonhypotensive hemorrhage in nephrectomized dogs. Infusion of AII (10 ng/kg.min) into a common carotid artery in nonbled dogs did not increase plasma ADH levels, suggesting that increases in carotid arterial plasma AII concentration alone do not stimulate an increase in ADH release. Subsequently, nephrectomized dogs subjected to nonhypotensive hemorrhage (0.44 ml/kg.min) were infused as follows: 0.9% saline intravenously, AII (10 ng/kg.min) intravenously, or AII (10 ng/kg.min) into the carotid. The Plasma ADH concentration increased in all three groups of dogs during hemorrhage. Although the AII-infused dogs demonstrated significant increases in plasma ADH levels earlier during hemorrhage, these changes were small; there were no statistically significant differences in plasma ADH concentrations among the three groups. These results suggest that increases in plasma AII concentration have little or no significant effect on the volume control of ADH release.