Sam Rebello

Effect of diaspirin crosslinked and stroma-reduced hemoglobin on mean arterial pressure and endothelin-1 concentration in rats

The effect of unmodified stroma reduced (SRHb) and modified diaspirin crosslinked (DCLHb) hemoglobin solutions on the mean arterial pressure and endothelin-1 (ET-1) concentration in blood plasma and various tissues was studied. Infusion of DCLHb or SRHb increased mean arterial blood pressure by 96% and 39%, respectively. Heart rate was not significantly affected by DCLHb or SRHb. A significant increase (P < 0.003) in the ET-1 levels in blood plasma after DCLHb and SRHb infusion was observed. The increase in plasma ET-1 concentration was significantly more marked with SRHb (141%) as compared to DCLHb (78%) treated rats. The concentration of ET-1 in the heart and brain regions was not altered in DCLHb or SRHb treated rats as compared to control. However, ET-1 concentration was significantly increased in the thoracic aorta (151%) and renal medulla (272%) of DCLHb treated rats. SRHb treated rats also showed a significant increase in ET-1 concentration in the thoracic aorta (141%) and renal medulla (429%). The effect of SRHb on the renal medulla was found to be significantly greater than that of DCLHb. ET may be one of the factors responsible for the cardiovascular effects of hemoglobin solutions.

Systemic hemodynamic and regional circulatory effects of centrally administered endothelin-1 are mediated through ETA receptors

Central endothelin (ET) has been implicated in the regulation of the cardiovascular system. The effect of intracerebroventricular (i.c.v.) administration of ET-1 or IRL 1620 (5, 15 and 45 ng) on the systemic hemodynamics and regional circulation was studied in anesthetized rats using a radioactive microsphere technique. Systemic hemodynamics and regional blood circulation were determined before (baseline) and at 30 min after the injection of each dose of ET-1 or IRL 1620. Administration of saline (5 microliters, i.c.v.) did not produce any significant cardiovascular effects. The lower doses of ET-1 (5 and 15 ng) did not produce any significant effect on blood pressure (BP), heart rate (HR), cardiac output (CO), stroke volume (SV), total peripheral resistance (TPR) and regional blood circulation. However, the higher dose (45 ng) produced a transient rise (26%) followed by a sustained fall (48%) in BP. The decrease in BP was accompanied by significant decreases in CO (44%) and SV (39%), while HR and TPR were not affected. ET-1 (45 ng, i.c.v.) also produced a significant reduction in blood flow to the brain (75%), heart (49%), kidneys (66%), GIT (40%), portal system (52%) and musculo-skeletal system (38%), while blood flow to the skin was not affected. To determine pharmacological specificity of the central effects of ET-1, studies were performed in rats pretreated with BQ-123, a specific ETA receptor antagonist. Pretreatment with BQ-123 (10 micrograms, i.c.v.), 15 min prior to the administration of ET-1, completely antagonized the systemic hemodynamic as well as the regional circulatory effects of ET-1 (45 ng, i.c.v.). In order to determine whether stimulation of central ETB receptors produces any cardiovascular effects, studies were performed using IRL 1620, a specific ETB receptor agonist. Administration of IRL 1620 (5, 15 and 45 ng, i.c.v.) did not produce any effect on systemic hemodynamics and regional blood circulation in rats. It is concluded that ETA but not ETB receptors are involved in the central cardiovascular actions of ET.

Regional circulatory and systemic hemodynamic effects of diaspirin cross-linked hemoglobin in the rat.

Diaspirin cross-linked hemoglobin (DCLHb) (Baxter Healthcare Corporation) is a promising resuscitative fluid. The effect of DCLHb (400 mg/kg, i.v.), on regional circulation and systemic hemodynamics was studied in male Sprague-Dawley rats using a radioactive microsphere technique. Systemic hemodynamics, distribution of cardiac output, regional blood flow and vascular resistance were determined before (baseline) and 15, 30 and 60 min after the administration of DCLHb. Infusion of an equal volume of saline did not produce any significant change in systemic hemodynamics or regional circulation. DCLHb produced an increase (79%) in the mean blood pressure which lasted for more than 60 min. Heart rate, cardiac output and stroke volume were not significantly affected, while total peripheral resistance was increased after the administration of DCLHb. DCLHb produced significant increases in blood flow to the heart, gastrointestinal tract (GIT), portal system and skin. The blood flow to the kidney, brain and musculoskeletal system was not significantly affected by DCLHb. The vascular resistance was not altered in the heart, brain, GIT, portal system, kidney or skin, but there was a marked increase in the vascular resistance in the musculoskeletal system. There was a significant increase in the percentage of cardiac output to visceral organs like heart, GIT and portal system, while a marked decrease in the percent cardiac output to musculoskeletal system was observed with DCLHb. It is concluded that the blood flow to most of the organs is either increased or is not affected by DCLHb.

Diaspirin Cross-Linked Hemoglobin (DCLHB TM): Involvement of Adrenergic Mechanisms in the Pressor Effect

Diaspirin cross-linked Hemoglobin (DCLHb) (400 mg/kg, i.v.), a resuscitative solution, produces a pressor effect in rats and several other species. Studies were conducted to determine the role of the central nervous system and adrenal medulla in the pressor effect of DCLHb in rats. Intravenous administration of DCLHb produced an increase in blood pressure in cervical sectioned animals, which was comparable to that observed in normal rats. This indicates that the pressor effect of DCLHb was mediated through the peripheral vascular system rather than through the central nervous system. DCLHb produced a pressor effect in bilateral adrenal demedullated rats that was similar to normal rats, suggesting that the pressor effect is not through the release of catecholamines or other pressor substance from the adrenal medulla. The effects of DCLHb pretreatment on norepinephrine (0.5 microgram/kg), phenylephrine (5 micrograms/kg) and clonidine induced blood pressure and heart rate responses were also studied. DCLHb significantly potentiated the pressor response to norepinephrine and phenylephrine. Clonidine normally produces a fall in blood pressure by acting on the central alpha-adrenoceptors, and a rise in blood pressure by stimulating the peripheral vascular alpha-adrenoceptors. DCLHb produced a marked potentiation of the pressor response to clonidine (75 micrograms/kg, i.v.), that masked the central depressor effect. The specificity of the potentiation was confirmed by using phenoxybenzamine, prazosin, and yohimbine. In order to exclude the contribution of a centrally induced cardiovascular effect of clonidine, further studies were carried out in cervical sectioned rats. DCLHb markedly potentiated the pressor effect of clonidine (25 micrograms/kg, i.v.) in cervical sectioned rats. This potentiation could be attenuated by prazosin and yohimbine.(ABSTRACT TRUNCATED AT 250 WORDS)

Down-regulation of endothelin receptors in the ventrolateral medulla of spontaneously hypertensive rats

The binding of [125I] sarafotoxin 6b (SRT 6b) and [125I] endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [125I] SRT 6b bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [125I] SRT 6b in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [125I] SRT 6b binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to decrease (48%) in the Bmax values in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. [125I] ET-1 also bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [125I] ET-1 in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [125I] ET-1 binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to 36% decrease in the Bmax values in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. It is concluded that the population of ET receptors is less in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.

CVS-1123, a direct thrombin inhibitor, prevents occlusive arterial and venous thrombosis in a canine model of vascular injury.

CVS-1123, low-molecular-weight, direct thrombin inhibitor was studied in an anesthetized canine model of arterial and venous thrombosis to determine whether thrombin inhibition could reduce the incidence of occlusive thrombosis in response to vessel-wall injury. The left carotid artery (LCA) and right jugular vein (RJV) were instrumented with a flow probe, intraluminal electrode, and critical stenosis. Either saline (n = 9), or CVS-1123 (n = 12) was administered in a loading dose of 2 mg/kg i.v., followed by an infusion (2.46 mg/kg/h for 180 min). Vessel-wall injury was initiated by applying a 300-microA anodal current to the intimal surface of the LCA and RJV. Platelet aggregation in response to gamma-thrombin remained inhibited by CVS-1123 for 8 h. The activated partial thromboplastin time (aPTT) was increased and remained elevated for the duration of the protocol. The prothrombin time (PT) showed an initial increase and then a rapid decrease after the infusion was discontinued. There was a twofold increase in the bleeding time (BT) at 2 h. The time to occlusion of the LCA was prolonged (380 +/- 22 min in the CVS-1123 group vs. 152 +/- 18 min in the saline group) with seven of 12 patent arteries at 8 h. Similarly, the time to occlusion for RJV was prolonged (415 +/- 16 min in the CVS-1123 group vs. 99 +/- 8 min in the saline group) with eight of 12 veins remaining patent at 8 h. CVS-1123 administration was associated with a decrease in the thrombus weights in both the LCA and RJV as compared with the saline-treated animals. In summary, CVS-1123 modifies the thrombogenic response to deep vessel-wall injury in both the arterial and venous circulations. The results suggest that CVS-1123 is an effective antithrombin and may offer a therapeutic alternative to current antithrombins in the management of arterial and venous thrombosis.

Changes in the Concentration of Endothelin-1 during Development of Hypertensive Rats

The concentration of endothelin-1 (ET-1) in the brain regions, heart, and throacic aorta of 1-, 4-, 6- and 8-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined using radioimmunoassay. ET-1-like immunoreactivity in the brain regions of 1-week-old WKY and SHR rats was lower compared to older (6 and 8 weeks) rats. ET-1 levels in the central nervous system gradually increased with age in both SHR and WKY rats. However, the concentration of ET-1 in 8-week-old rats was lower in the brain regions of SHR compared to WKY rats. The concentration of ET-1 in the thoracic aorta of SHR (224 +/- 43 pg/g tissue) rats was lower than that of WKY (452 +/- 11 pg/g tissue) rats at 1 week of age. However, ET-1 levels gradually increased with age in SHR rats. By 8 weeks of age, levels of ET-1 in SHR (623 +/- 33 pg/g tissue) rats were higher compared to WKY (439 +/- 62 pg/g tissue) rats. In the heart, ET-1 levels were similar in WKY and SHR rats at 4 weeks of age, but at 8 weeks of age ET-1 levels were higher in SHR rats (364 +/- 33 pg/g tissue) compared to WKY rats (260 +/- 31 pg/g tissue). It appears that at 8 weeks of age when hypertension is fully expressed in rats, ET-1 levels are lower in the central nervous system and are higher in the thoracic aorta and heart of SHR compared to WKY rats.

Cardiovascular effects of centrally administered endothelin-1 in rats.

Summary: The effect of centrally administered endothelin-1 (ET-1) or IRL 1620 (5, 15, and 45 ng) on systemic hemodynamics was studied in anesthetized rats using a radioactive microsphere technique. Administration of saline (5 μ, i.c.v.) did not product any significant cardiovascular effects. The lower doses of ET-1 (5 and 15 ng) did not produce any significant effect on blood pressure (BP), heart rate (HR), cardiac output (CO), stroke volume (SV), and total peripheral resistance (TPR). However, the highest dose (45 ng) produced a transient rise followed by a sustained fall in BP. The CO and SV decreased significantly, whereas HR and TPR were not affected. Pretreatment with BQ-123 (10 μg, i.c.v.) 15 min before administration of ET-1, completely antagonized the systemic hemodynamic effects of ET-1. Centrally administered IRL 1620, a specific ETB receptor agonist, did not produce any effect on BP, HR, CO, SV, and TPR in rats. It is concluded that ETA but not ETB receptors are involved in the central cardiovascular actions of ET.

Characteristics of endothelin receptors in the cerebral cortex and spinal cord of aged rats

Characteristics of endothelin receptors were studied in male Fischer 344 rats at 4-, 15- and 24-months of age and [125I]ET-1 binding showed a single high affinity binding site in their cerebral cortex and spinal cord membranes. The density and affinity of ET binding sites were found to be similar in rats of various age groups. To determine the affinity of ET isotypes to ET-1 binding sites in cerebral cortex and spinal cord, competition studies were performed and K(i) values of ET-1, ET-2 and ET-3 for [125I] ET-1 binding sites were determined. It was found that ET-1 had 100 and ET-2 had 25-100 times lower K(i) values as compared to ET-3, indicating that ET receptors in cerebral cortex and spinal cord are of ETA type. In spinal cord, the K(i) values of ET-1 and ET-2 for ET receptor were found to be similar. However, in cerebral cortex the K(i) values of ET-1 were found to be at least 6 times lower than ET-2. It is inferred that there are two subtypes of ET(A) receptors, ET(A1) which have higher affinity for ET-1 in comparison to ET-2, as found in cerebral cortex and ET(A2) which have higher affinity for ET-2, as found in spinal cord. The K(i) and IC50 values of ET-1, ET-2, and ET-3 for [125I]ET-1 binding sites in cerebral cortex and spinal cord were found to be similar in 4-, 15- and 24-month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of endothelin and its receptors in rat brain

The ontogeny of endothelin (ET) system in rats was studied in preterm (18 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (Kd) and density (Bmax) were determined using [125I] ET-1 and [125I] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 +/- 3.18 pg/g protein in preterm, 47.37 +/- 5.31 pg/g protein in term and 48.30 +/- 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [125I] ET-1 and [125I] SRT 6b. The Kd values for [125I] ET-1 and [125I] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [125I] ET-1 and [125I] SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bmax values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density.