Martha L. Blair

Effects of angiotensin II on membrane current in cardiac Purkinje fibers.

Abstract We studied the actions of the octapeptide hormone angiotensin II (AII) on isolated cardiac Purkinje fibers. AII (1 to 75 n m ) increased the height and duration of the plateau phase of the action potential and increased the strength of contraction in these preparations. These effects were not blocked by propranolol (10 −6 m ). A two-microelectrode voltage clamp technique combined with simultaneous tension measurements was used to study the AII-induced changes in membrane current and contractile activation. AII enhanced peak tension and promoted an inward shift in the net membrane current-voltage relation at test voltages between −40 and 0 mV. The inward shift in current was maintained for the duration of the 500 ms test voltage step. The AII-induced current shift was reduced or abolished when external calcium concentration was decreased from 5.4 m m to 1.8 m m , and was inhibited by the calcium antagonist D600.

Vasopressin and renin response to dehydration in aged rats

Abnormalities in neurohypophyseal function have been postulated to contribute to the alterations in fluid and electrolyte balance observed during aging. In this study, parameters of fluid and electrolyte balance were evaluated during chronic water deprivation in old (30 months) and young (3 months) Fischer 344 rats. The increase in serum vasopressin (VP) and renin concentrations observed in the 3 month animals following chronic water deprivation were absent in the aged rats (p less than 0.05 and p less than 0.02, respectively). This occurred in spite of apparently comparable alterations in fluid volume and osmolality (assessed by changes in body weight, hematocrit and plasma osmolality). Relative to body weight, VP content of the neural lobe was significantly reduced and was more severely depleted by dehydration in aged rats than in young rats. Thus, inadequate neurohypophyseal hormone stores may contribute to the inability of the aged animals to attain elevated serum VP concentrations during chronic stimulation. Several parameters of renal function were examined in the aged rats. Although none of the old rats were in renal failure, they all showed some indication of reduced renal function. In spite of renal abnormalities including reduced concentrating capabilities, the old rats did demonstrate a significant antidiuretic response to dehydration. However, with prolonged fluid deprivation, they were unable to attain serum VP or renin concentrations comparable to that achieved by the young rats.

Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulation using P-selectin in an implantable device

Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously, we have demonstrated an ability to mimic physiological cell trafficking in order to capture CD34‐positive (CD34+) HSPCs using monolayers of the cell adhesion protein P‐selectin in flow chambers. The current study aimed to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P‐selectin, were inserted into the femoral artery of rats. Blood flow through the cell capture device resulted in a wall shear stress of 4–6 dynes/cm2. After 1‐h blood perfusion, immunofluorescence microscopy and flow cytometric analysis revealed successful capture of mononuclear cells positive for the HSPC surface marker CD34. Purity of captured CD34+ cells showed sevenfold enrichment over levels found in whole blood, with an average purity of 28%. Robust cell capture and HSPC enrichment were also demonstrated in devices that were implanted in a closed‐loop arterio‐venous shunt conformation for 2 h. Adherent cells were viable in culture and able to differentiate into burst‐forming units. This study demonstrated an ability to mimic the physiological arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.

ROLE OF THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS IN CARDIOVASCULAR REGULATION

1. The paraventricular hypothalamic nucleus (PVH) is a complex structure with both neuroendocrine and autonomic functions. It is a major source of vasopressin and the primary source of corticotropin‐releasing factor. In addition, parvicellular PVH neurons have reciprocal connections with brain‐stem autonomic centres and directly innervate sympathetic preganglionic neurons. Evidence is reviewed which indicates that in conscious rats PVH activation increases blood pressure, heart rate, renal nerve activity and plasma renin activity.

Lesion of the area postrema region attenuates hypertension in spontaneously hypertensive rats.

To determine whether the area postrema contributes to the development of hypertension in spontaneously hypertensive rats (SHR), sham or electrolytic lesions of the area postrema (AP) were made in 4-week-old SHR and Wistar-Kyoto (WKY) controls. From weeks 5 through 16, systolic pressure was measured via tail plethysmography. While blood pressure rose markedly in sham-operated SHR, increases in pressure were small in AP-ablated SHR and similar to those seen in all WKY. Subsequent direct measurements of mean arterial pressure in the same rats showed a significant correlation (r=0.87, p<0.01) with the pressure data acquired via weekly tail-cuff measurement, thereby confirming that hypertension in AP-ablated SHR had indeed been attenuated. Analysis of several hundred computer-acquired measurements of mean arterial pressure from each rat showed that AP ablation shifted the distribution of mean arterial pressure to a lower range in SHR but not WKY. Ablation of the AP also decreased resting heart rate in SHR but not WKY. Suppression of heart rate in response to intravenous phenylephrine was equivalent in sham-operated and AP-ablated rats, suggesting that baroreflex-mediated slowing of heart rate was not impaired. In response to intravenous angiotensin II, suppression of heart rate was similar in sham and AP-ablated SHR, and actually was enhanced in AP-ablated WKY. Histological evaluation of the lesions indicated that visible damage to the adjacent nuclei of the solitary tracts was confined to a small portion of the commissurel nucleus. Although we cannot rule out the possibility of damage to the remaining nuclei of the solitary tracts, gross functional damage (which would be revealed by increased lability of arterial pressure and/or decreased baroreflex sensitivity) was undetectable in the baroreflex and lability data. We conclude that ablation of the area postrema region markedly attenuates the development of hypertension in the SHR model.

Attenuation of spontaneous hypertension in rats by a vasopressin antagonist.

Although abnormalities in the vasopressin system have been reported in spontaneously hypertensive rats (SHR), neither short-term nor long-term administration of the vasopressin antagonist d(CH2)5-Tyr(Me)arginine vasopressin (AVP), which selectively blocks the action of vasopressin on vascular (V1) receptors, altered the course of hypertension in SHR. In the current study, long-term administration of a different vasopressin antagonist, d(CH2)5-D-Tyr(Me)VAVP, to SHR and Wistar-Kyoto rats (WKY) from 4 to 12 weeks of age significantly attenuated the development of systolic hypertension in SHR (p < 0.05) without altering Mood pressure in normotensive WKY. The antagonist was delivered subcutaneously by osmopump at 0.1 μ Systolic blood pressure was monitored twice weekly by tail plethysmography beginning at 5 weeks of age. In a second group of SHR, the drug infusion was continued until 18 weeks of age. In this group, the attenuation of systolic hypertension by the drug was extended and became more prominent (p < 0.007). Resting mean arterial pressure measured by Indwelling catheters in the conscious state at 18 weeks of age was significantly reduced in the antagonist-treated SHR (144 ± 4 vs 157 ± 4 mm Hg;p < 0.05). Heart rate also was significantly reduced by the drug (351 ± 6 vs 392 ± 7 beats/min; p < 0.001). Following measurement of mean arterial pressure in the rats at 18 weeks of age, the osmopumps were removed and systolic blood pressure, mean arterial pressure, and heart rate were observed until 22 weeks of age. All of these parameters returned to the levels observed in untreated SHR within 2 weeks after drug withdrawal. Although this antagonist has both V, and V2 (antidiuretlc) antagonist properties, the infusion protocol used in this study resulted in antagonism of the pressor action of vasopressin but incomplete antagonism of the antldluretk action of vasopressin. Thus, the mechanism responsible for the antihypertensive action of this antagonist is not clear, but the results suggest that long-term blockade of the actions of endogenous vasopressin does alter the course of hypertension in SHR.

Evidence against a pressor role for vasopressin in spontaneous hypertension.

The hypothesis that the vasoconstrictor action of vasopressin may contribute to the development of hypertension in spontaneously hypertensive rats (SHR) was tested by chronic infusion of a specific antagonist of the vascular effects of vasopressin. From 4 to 13 weeks of age, SHR and Wistar-Kyoto rats (WKY) received subcutaneously either isotonic saline or the vasopressin pressor antagonist, d(CH2)5Tyr(Me)arginine vasopressin by osmopump. Systolic blood pressure was measured by tail cuff from 5 to 11 weeks of age. In SHR, the vasopressin analogue did not alter the rate or magnitude of increase in systolic blood pressure. In WKY, systolic blood pressure in the vasopressin analogue group was slightly reduced compared with the saline infusion values until 10 weeks of age (F1, 10 = 10.18, p = 0.008). At 12 to 14 weeks of age, all animals were prepared with indwelling arterial and venous catheters. Resting mean arterial pressure was not altered significantly by the vasopressin analogue infusion in either strain, but the response to an acute vasopressin infusion of 5, 15, or 50 ng/kg body weight was markedly attenuated by the analogue treatment, indicating that plasma levels of the vasopressin analogue were sufficient to block pressor effects of endogenous vasopressin. A bolus injection of the angiotensin II converting enzyme inhibitor teprotide (SQ 20881) resulted in a decrease in mean arterial pressure (p less than 0.05) that was comparable in all groups, and serum renin concentration was not elevated in the vasopressin analogue-treated rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic stimulation of vasopressin release in spontaneously hypertensive rats.

Plasma vasopressin (VP) concentration is elevated in spontaneously hypertensive rats (SHRs) relative to their normotensive Wistar-Kyoto (WKY) controls. The possibility that this reflects altered responsiveness of the hypothalamo-neurohypophyseal system (HNS) in SHRs was examined by comparing VP release in response to acetylcholine from organ cultured HNS explants obtained from SHR and WKY donors. Explants were prepared from 5-, 8-, and 18-week-old animals. Blood pressure was significantly elevated in the 8- and 18-week-old SHR donors relative to their age-matched WKY donors. VP release was assessed on the 4th day of culture during a control hour and during the subsequent hour in the presence of acetylcholine. Acetylcholine caused a concentration-dependent stimulation of VP release from both types of explants, but the response was significantly greater in the explants from 5- and 8-week-old SHRs than in explants from age-matched WKYs. The explants from 18-week-old SHRs and WKYs demonstrated comparable sensitivity to acetylcholine. Basal VP release was not significantly different in explants from age-matched SHRs and WKYs, but it did increase with donor age in both strains. These studies indicate potential hyperresponsiveness of the HNS to excitatory stimuli in SHRs during the developmental phase of hypertension. The hyperresponsiveness disappears in the chronically hypertensive phase. Thus, increased sensitivity of the HNS during the development of hypertension may contribute to the elevation of plasma VP concentration in SHRs.

Abnormalities in hypothalamic and neurohypophysial vasopressin content are not a consequence of hypertension in the spontaneously hypertensive rat

In order to determine if the decreased hypothalamic and increased posterior pituitary content of vasopressin (VP) observed previously in spontaneously hypertensive rats (SHR) were a secondary consequence of the hypertension, the effect of preventing the development of hypertension on VP content of the hypothalamoneurohypophyseal system was evaluated. Two methods for preventing the hypertension were used: (1) chronic angiotensin-converting enzyme inhibition (oral captopril, 100 mg/kg/day at 4-12 weeks of age); and (2) intraventricular 6-hydroxydopamine (6-OHDA, 200 micrograms at 4 and 5 weeks of age). Both of these treatments markedly attenuated the increase in systolic blood pressure in SHRs at 5-11 weeks of age. The captopril-treated rats had a significant elevation in serum renin activity at 12 weeks of age indicating the presence of chronic converting enzyme inhibition, and the 6-OHDA-treatment resulted in a depletion of hypothalamic (86%) and brainstem (76%) norepinephrine content. Hypothalamic VP content was reduced in untreated SHRs compared to normotensive Wistar-Kyoto rats (WKYs, P = 0.0015). It was not significantly altered in either strain by the 6-OHDA treatment. Captopril caused a reduction in hypothalamic VP content in both SHRs and WKYs (P less than 0.01). Posterior pituitary VP content was elevated in untreated SHRs compared to WKYs (P less than 0.001), and remained elevated with captopril and 6-OHDA treatments. These data indicate that the abnormalities in VP content in the hypothalamus and posterior pituitary of SHRs are not a response to the hypertension. Therefore, they may represent primary abnormalities in the SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditioned increase in peripheral blood mononuclear cell (PBMC) number and corticosterone secretion in the rat

Femoral artery catheters were surgically implanted into male Lewis/N rats to allow blood sampling and drug infusion in the freely moving animal. After recovery, conditioned animals received four pairings of a peppermint odor, the conditioned stimulus (CS), and an infusion of 0.1 mg/kg nicotine bitartrate, an unconditioned stimulus (US) for an increase in the number of peripheral blood mononuclear cells (PBMC) and an increase in corticosterone concentration. When reexposed to the peppermint odor, conditioned animals showed a significant increase in PBMC number and corticosterone secretion when compared to saline and unpaired control groups and previously conditioned animals that were not reexposed to the CS. Increased PBMCs were found on the fifth unreinforced CS trial. Conditioned CORT responses were lost after the initial test trial. The data indicate that the distribution of immune cells can be influenced by learning processes and support the role of learning in the regulation of corticosterone secretion.