The renin-angiotensin system in nonmammalian vertebrates.

The most primitive components of the RAS appeared early in the phylogenetic history of vertebrate animals. It is probable that renin granules were present in the kidneys of ancestral chordates before divergence in the evolution of actinopterygian fish and tetrapods occurred. Granulated juxtaglomerular cells similar to the renin-containing cells of the mammalian nephron are found in most extant vertebrate species although not in agnathan and elasmobranch fish. A macula densa occurs in amphibians, birds and mammals; and an extraglomerular mesangium, only in birds and mammals. Renin-like activity and angiotensin-like pressor material have been demonstrated in all classes of vertebrates. The amino acid sequences of native ANG I have been determined for representative species of teleost fish, amphibian, reptile and bird. These peptides differ from mammalian angiotensins at positions 1, 5 and 9. The RAS appears to be involved in osmoregulation, ionoregulation and the control of blood circulation. Prolonged hypovolemic hypotension or sodium depletion increases renin levels. Angiotensins elicit drinking and stimulate transepithelial ion transport. However, direct steroidogenic and antidiuretic hormone-releasing activities, which would promote mineral and fluid conservation, have not been demonstrated unambiguously in nonmammalian vertebrates. ANG II raises blood pressure by direct vasoconstrictor action on arteriolar muscles in some animals, but perhaps more generally by acting on the nervous system and adrenal paraneurons. In birds the hormone also has a hypotensive effect. ANG II stimulates the SNS in agnathans, elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. Thus, modulation of sympathetic activity may be one of the most primitive and conservative functions of the RAS. For this reason, nonmammalian vertebrates are valuable models for studying the neurogenic actions of angiotensin II relevant to hypertensive disease.

Adaptive regulation of ascorbate transport in osteoblastic cells.

Osteoblasts possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent ascorbate transporter located in the plasma membrane. The transporter is specific for ascorbate and stereoselective for L-ascorbate over D-isoascorbate. The present study examined the effects of ascorbate supplementation and deprivation on the activity of this transport system. L-ascorbate transport activity was determined by measuring uptake of the vitamin by ROS 17/2.8 osteosarcoma cells during 1 minute incubations with 5 microM L-[14C]ascorbate. The initial rate of L-[14C]ascorbate uptake by ROS 17/2.8 cells grown for 18 h in L-ascorbate-replete medium was 89 +/- 8 nmol/g protein per minute. Following removal of L-ascorbate from the growth medium, the initial rate of uptake increased within 6 h to 126 +/- 13 nmol/g protein per minute. Conversely, the initial rate of uptake by cells grown in ascorbate-free medium decreased following the addition of L-ascorbate, but not D-isoascorbate, to the medium. The effect of ascorbate pretreatment was specific for ascorbate transport in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-glucose. Kinetic analysis revealed that modulation of ascorbate transport arose from changes in the apparent maximum rate of transport (Vmax) without changes in the affinity of the transport system for L-ascorbate. These experiments are the first to show that ascorbate transport by osteoblastic cells responds to vitamin C deprivation and supplementation. Adaptation of transport activity to substrate availability may play an important role in the physiological regulation of intracellular ascorbate levels.

Ascorbate uptake by ROS 17/2.8 osteoblast-like cells: substrate specificity and sensitivity to transport inhibitors.

Ascorbate (reduced vitamin C) is required for bone formation. We have shown previously that both the osteoblast-like cell line ROS 17/2.8 and primary cultures of rat calvarial cells possess a saturable, Na(+)-dependent uptake system for L-ascorbate (J Membr Biol 111:83-91, 1989). The purpose of the present study was to investigate the specificity of this transport system for organic anions and its sensitivity to transport inhibitors. Initial rates of ascorbate uptake were measured by incubating ROS 17/2.8 cells with [L-14C]ascorbate at 37 degrees C. Uptake of [L-14C]ascorbate (5 microM) was inhibited 98 +/- 1% by coincubation with unlabeled L-ascorbate (3 mM) and 48 +/- 4% by salicylate (3 mM), but it was not affected by 3 mM formate, lactate, pyruvate, gluconate, oxalate, malonate, or succinate. Uptake of the radiolabeled vitamin also was not affected by acute (1 minute) exposure of the cells to the Na+ transport inhibitors amiloride and ouabain or the glucose transport inhibitor cytochalasin B. In contrast, anion transport inhibitors rapidly (less than 1 minute) and reversibly blocked [L-14C]ascorbate uptake. In order of potency, these drugs were 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) approximately equal to sulfinpyrazone greater than furosemide approximately equal to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). These findings indicate that the ascorbate transporter is relatively specific for the ascorbate anion, since other organic anions (with the exception of salicylate) did not compete with ascorbate for uptake. Rapid and reversible inhibition by the impermeant antagonists DIDS and SITS suggests that they interact directly with the ascorbate transporter, consistent with location of the transport system in the plasma membrane.

Transforming growth factor-beta stimulates ascorbate transport activity in osteoblastic cells.

Transforming growth factor-beta (TGF beta) modulates the proliferation and differentiation of a number of cell types, including osteoblasts. TGF beta has been shown to stimulate matrix synthesis by connective tissue cells, but its mechanism of action is poorly understood. Because ascorbate (reduced vitamin C) also influences osteoblastic differentiation and is required as a cofactor for collagen synthesis, the present study examined the effect of TGF beta on osteoblastic ascorbate uptake. Saturable Na(+)-dependent uptake of ascorbate by cultures of UMR-106 rat osteosarcoma cells proceeded linearly with time for at least 10 min at 37 C. Exposure of cultures to TGF beta 1 stimulated initial rates of saturable Na(+)-dependent ascorbate transport, but did not affect nonspecific uptake or binding of the vitamin. Cells pretreated for 24 h with either vehicle or TGF beta 1 (3 ng/ml) and then assayed for transport of L-[14C] ascorbate (10 microM) showed significantly different transport activities (vehicle, 30 +/- 2; TGF beta 1, 44 +/- 3 nmol ascorbate/g protein/min; n = 14; P less than 0.005). Kinetic studies revealed that TGF beta 1 increased the maximum velocity of ascorbate transport without changing the affinity of the transporter for the vitamin, since the apparent maximum velocity increased from 83 to 106 nmol ascorbate/g protein/min; while the apparent Km remained unchanged at 20 microM L-ascorbate. The effect of this growth factor on ascorbate transport appeared to require protein synthesis, because it was completely blocked by cycloheximide. These results are consistent with TGF beta 1 increasing the rate of synthesis of either new Na+ ascorbate cotransporters or a regulatory protein that interacts with existing transporters to increase their turnover number. Enhanced uptake of ascorbate may contribute to the increase in collagen synthesis induced by TGF beta.

6-Hydroxydopamine treatment diminishes noradrenergic and pressor responses to angiotensin II in adrenalectomized ducks.

The extent to which the vasopressor action of angiotensin II (AII) in birds depends upon the sympathetic nervous system in not known. Therefore, we have tested whether the sympathetic neurotransmitter norepinephrine (NE) mediates the residual vasopressor response to AII in adrenalectomized ducks (Anas platyrhynchos). Treatment with 6-hydroxydopamine (6-OHDA) inhibited by up to 100% the pressor effect of exogenous AII in anesthetized adrenalectomized ducks. After high doses of AII, a transient drop in blood pressure (BP) often preceded or supplanted the pressor response. Systolic BP responses to injections of tyramine also were decreased by 6-OHDA, but responses to NE were increased (diastolic BP) or not affected significantly (systolic BP). Inhibition with SQ20881 of converting enzyme, to decrease endogenous production of AII, reduced pressor responses to AI, but did not affect either baseline BP or responses to AII. Thus, the diminished responsiveness to AII in these catecholamine-depleted ducks was not due to competition from endogeneous AII. Furthermore, the increases in plasma NE and E concentrations that normally follow AII injection were abolished by adrenalectomy and 6-OHDA treatment. The coincident extinction of both cathecholamine and vasopressor responses comprises new evidence that AII raises BP in the Pekin duck by stimulating the sympathetic nervous system. The vasopressor response is mediated by extraadrenal as well as adrenal sources of NE.

Adrenalectomy inhibits noradrenergic, adrenergic, and vasopressor responses to angiotensin II in the Pekin duck (Anas platyrhynchos).

The iv injection of Asp1, Val5-angiotensin II(AII) was followed by increases in arterial plasma norepinephrine (NE) and epinephrine (E) concentrations and elevation of arterial blood pressure in the anesthetized Pekin duck. AII injection did not affect the concentration of unconjugated dopamine in arterial plasma. Adrenalectomy inhibited the increases in plasma NE and E concentrations elicited by AII. Pressor responses to AII also were diminished after adrenalectomy, whereas pressor responses to exogenous NE and tyramine were not measurably affected. The alpha-adrenergic blocking drug phenoxybenzamine diminished the pressor effect of AII in both shamoperated and adrenalectomized ducks. The blockade of alpha-adrenergic receptors by phenoxybenzamine was incomplete, since pressor responses to exogenous NE were lessened by not abolished. We conclude from these experiments that AII increases arterial blood pressure in the Pekin duck by mobilizing NE and E from both adrenal and extraadrenal stores. It remains unknown whether the residual pressor response to AII in phenoxybenzamine-treated, adrenalectomized ducks involves an alpha-adrenergic mechanism.

Inhibitory actions of calmodulin antagonists on steroidogenesis in zona glomerulosa cells.

The stimulation of aldosterone (Aldo) production in the adrenal glomerulosa cell by angiotensin II (AII) and ACTH is highly dependent on calcium. To determine the role of calmodulin (CM) as a regulator of intracellular calcium during hormonal activation of steroidogenesis, the actions of a series of CM inhibitors on the stimulation of Aldo biosynthesis by AII, the calcium ionophore A23187, ACTH, and cAMP were examined in isolated adrenal glomerulosa cells. The CM antagonists, pimozide, W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and W5 [N-(6-aminohexyl)-1-napthalenesulfonamide] caused dose-dependent inhibition of hormone-stimulated Aldo production, in proportion to their reported binding affinities for CM. The maximum inhibitory concentration (5 microM) of the most potent CM antagonist, pimozide, had no effect on the conversion of progesterone to Aldo. However, submaximal inhibitory concentrations of W7 and W5 also reduced conversion of progesterone to Aldo, indicating that their inhibitory action is not only due to their CM antagonist properties. The relative sensitivities of the stimuli of Aldo release to inhibition by all three CM inhibitors were A23187 = AII greater than ACTH greater than 8-bromo-cAMP (8-Br-cAMP). Aldo responses to ACTH were more sensitive to inhibition by pimozide than those to 8-Br-cAMP, suggesting that ACTH-induced cAMP formation is also dependent on CM. Pimozide (5 microM) completely inhibited the Aldo responses to AII and A23187, whereas the Aldo responses to ACTH and 8-Br-cAMP were only partially inhibited. At submaximal inhibitory concentrations, pimozide caused a decrease in the maximum response to AII without changing the concentration of the peptide required for half-maximum stimulation of Aldo production. The inhibition of Aldo production by pimozide was accompanied by parallel inhibition of pregnenolone accumulation measured in the presence of cyanoketone. No effect of pimozide (5 microM) was observed on the binding of 125I-AII to adrenal glomerulosa cells, placing the CM-dependent site between the receptor and the side-chain cleavage enzyme. These findings indicate that CM-mediated regulation is essential for the steroidogenic response to calcium ionophore and AII, but is only partially required for the responses to ACTH and 8-Br-cAMP.

Insulin stimulates vitamin C recycling and ascorbate accumulation in osteoblastic cells.

Insulin modulates the differentiation and synthetic activity of osteoblasts, but its mechanisms of action are not fully understood. Because ascorbate also influences osteoblast differentiation and is a cofactor for collagen synthesis, we examined the effects of insulin on the transport and metabolism of vitamin C in osteoblastic cells. UMR-106 rat osteoblast-like cells accumulated ascorbate intracellularly when incubated with dehydroascorbic acid (DHAA; oxidized vitamin C). Insulin increased the intracellular concentration of ascorbate derived from DHAA and also increased the initial rates of uptake of DHAA and 2-deoxyglucose, but not that of ascorbate. A half-maximal effect on DHAA uptake was observed with approximately 100 pM insulin, whereas insulin-like growth factor I (IGF-I) was less potent. Preincubation with insulin for 6-12 h was required for stimulation, similar to the period needed for increased expression of facilitative hexose transporters (GLUT). DHAA uptake was inhibited by the GLUT antagonist cytochalasin B as well as by the GLUT substrates D-glucose and 2-deoxyglucose, whereas L-glucose and fructose had no effect. We conclude that insulin and IGF-I stimulate osteoblastic uptake of DHAA through facilitative hexose transporters. The relative potency of insulin in stimulating DHAA uptake is consistent with mediation by insulin receptors. DHAA is reduced to ascorbate within osteoblasts, maintaining a high intracellular concentration of ascorbate available for collagen synthesis. Impaired uptake of DHAA may contribute to the osteopenia associated with type I diabetes. In addition, cytotoxic levels of DHAA may accumulate in the extracellular fluid due to decreased transport activity and competitive inhibition by elevated concentrations of glucose.

Angiotensin and converting enzyme regulate extrarenal salt excretion in ducks.

Numerous previous studies have proposed a salt-conserving role for the renin-angiotensin system in mammals, but there is little evidence of this putative role in birds. Especially interesting are marine birds, which have a relatively limited ability to regulate the osmolality and ionic composition of their urine but possess extrarenal salt glands capable of excreting a highly concentrated NaCl solution. In the present experiments, hypertonic saline, angiotensin I (ANG I), and captopril were infused iv into chronically cannulated ducks to study the neuroendocrine, cardiovascular, renal, and extrarenal excretory responses to osmotic stress. Infusion of hypertonic saline elicited nasal salt excretion, which could be stopped completely by coadministration of ANG I. The effective dose of ANG I increased the plasma norepinephrine (NE) concentration, but did not alter heart rate or arterial blood pressure. Captopril enhanced extrarenal salt excretion in the saline-loaded ducks. The converting enzyme inhibitor also blocked the noradrenergic and NaCl-retaining actions of ANG I; conversely, coadministration of captopril and ANG I increased the plasma epinephrine (E) concentration. These findings indicate that the renin-angiotensin system, in addition to effects on the sympathoadrenal system, regulates NaCl and water metabolism in birds with extrarenal salt glands.

Effect of age on autonomic and cardiac responses in a rat stroke model.

The cardiovascular system and its responses change with increasing age. This has seldom been considered in experimental models of stroke, although most strokes occur in the elderly. We studied 57 male Wistar rats in three age groups: 47 to 70 days old (juvenile), 110 to 152 days old (young adult), and 186 to 245 days old (mature adult), each group being subdivided into experimental and sham operation groups. All rats underwent occlusion or sham occlusion of the left middle cerebral artery and monitoring of the mean arterial blood pressure, heart rate, sympathetic nerve activity, plasma catecholamine levels, and electrocardiogram. Eight of the 12 rats in the oldest group died within 6 hours of the middle cerebral artery occlusion; of these, the youngest was 186 days old. The mature adult rats that died before completion of the experiment showed the highest level of sympathetic nerve activity and the only significant increase in the QT interval of the electrocardiogram. Following middle cerebral artery occlusion, sympathetic nerve activity increased in the young adult rats but most strikingly in the mature adult rats that died before the end of the 6-hour experiments. Plasma norepinephrine levels were significantly elevated at 4 and 6 hours after middle cerebral artery occlusion in the oldest group and only at 6 hours in the juvenile rats. The results of this study are consistent with impaired sympathetic and cardiovascular regulation in the mature adult rat. High sympathetic activity may represent one mechanism leading to fatal cardiac arrhythmias. Age-related impairment of sympathetic regulation may contribute to the higher mortality seen among elderly patients with stroke.

Asymmetry of sympathetic consequences of experimental stroke.

Asymmetries of sympathetic regulation at the level of the inferior cervical ganglia have long been recognized. Lateralization of autonomic representation may also occur in the brain, since inactivation of the left and right hemispheres by intracarotid amobarbital produces an increase and decrease in heart rate, respectively. However, this conclusion has remained tentative, since the differential effect of lateralized brain lesions on sympathetic activity has not been studied systematically. Forty-eight urethan-anesthetized Wistar rats were divided into three groups: a group given left middle cerebral artery occlusion, and a group given sham operation. Heart rate, mean arterial blood pressure, renal sympathetic nerve discharge, and electrocardiogram were monitored throughout the 4-hour experiments. Plasma epinephrine and norepinephrine levels were measured at baseline and 1 and 4 hours after occlusion or sham occlusion. The mean arterial pressure decreased in the group given sham operation and to lesser extent in the group given left middle cerebral artery occlusion. By contrast, mean arterial pressure did not fall in the group given right middle cerebral artery occlusion and at 4 hours was significantly higher than control values in the sham-occluded rats. Renal sympathetic nerve discharge was decreased in the sham-occluded group, increased significantly from 20 minutes to 2 hours in the group given left middle cerebral artery occlusion, and increased from about 20 minutes to the end of the experiment in the group given right middle cerebral artery occlusion. The plasma norepinephrine level was significantly elevated at 1 hour (93%) and 4 hours (44%) only in the group given right middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental profiles of antioxidant enzymes and trace metals in chick embryo.

It has been previously well documented that partial pressure of oxygen (PO2) and weight-specific rate of O2 consumption in chick embryo (Gallus gallus domesticus) transiently increase midway through the 21-day in ovo incubation period. The present study found that these oxidative changes were paralleled by the concentrations of glutathione (GSH) and Zn in liver and by the specific activity of superoxide dismutase (SOD) in brain. Levels of antioxidant enzymes and their trace metal cofactors were markedly higher in liver than in brain. Hepatic catalase activity changed in parallel with the concentration of its cofactor, Fe. However, the relative abundance of metal cofactors did not appear to be the determining influence on other antioxidant enzyme activities. Rates of extra-mitochondrial hydrogen peroxide release were also much greater in liver than in brain. Taken together, the results of this initial study of embryonic chick antioxidant systems suggest that certain antioxidants may be regulated by PO2 and rate of oxidative metabolism during fetal development.

Conjugated catecholamines and pressor responses to angiotensin, luteinizing hormone-releasing hormone and prazosin in conscious toads.

Synthetic angiotensin II (Ang II), mammalian luteinizing hormone-releasing hormone (LHRH) and salmon LHRH (sLHRH) were injected intravenously into conscious, adult toads (Bufo marinus) to elucidate the cardiovascular actions of the hormones. The maximal increases in pulse pressure elicited by the three peptides did not differ from each other but only Ang II increased cardiac frequency. The maximal increases in mean arterial blood pressure (MAP) caused by LHRH and sLHRH were identical, while Ang II caused a 100% greater maximal effect. The median effective doses (ED50) for both Ang II and LHRH were approximately 0.1 nmol kg-1 whereas the potency of sLHRH was 10 fold less. Pressor responses to LHRH and sLHRH were blocked completely by (D-pGlu1, D-Phe2, D-Trp3,6)-LHRH but this antagonist did not inhibit Ang II. Significant proportions of circulating, endogenous dopamine, noradrenaline (NA) and adrenaline (Ad) were found to be sulphoconjugated. Arterial plasma concentration of free NA increased simultaneously with the rise in blood pressure following Ang II injection. The magnitude of the free NA response increased with increasing Ang II dose but even a high dose failed to augment the plasma level of conjugated NA. Ang II did not alter concentrations of free or conjugated dopamine and Ad. Intraarterial injection of an alpha-adrenoceptor antagonist, prazosin, caused sustained elevation of arterial pressure and free Ad. Subsequently Ang II lowered plasma Ad concentration. Prazosin inhibited the NA response to Ang II yet the pressor effects of the alpha-adrenoceptor antagonist and Ang II were additive. Administration of a beta-adrenoceptor antagonist, propranolol, largely reversed the cardiovascular sequelae of alpha-adrenoceptor blockade. It is concluded, firstly, that the cardiovascular actions of Ang II and LHRH are mediated through different receptors. Secondly, although it had been shown that alpha- and beta-adrenoceptor mechanisms mediate the pressor effect of LHRH, the present experiments showed that mobilization of catecholamines cannot account for the pressor response to Ang II. Thirdly, both free and conjugated catecholamines circulate in toads; however the extent of conjugation can be dissociated from the changes in free NA and Ad induced by Ang II and prazosin.

Ascorbate prevents microvascular dysfunction in the skeletal muscle of the septic rat.

Septic patients have low plasma ascorbate concentrations and compromised microvascular perfusion. The purpose of the present experiments was to determine whether ascorbate improves capillary function in volume-resuscitated sepsis. Cecal ligation and perforation (CLP) was performed on male Sprague-Dawley rats. The concentration of ascorbate in plasma and urine, mean arterial blood pressure, and density of continuously perfused capillaries in the extensor digitorum longus muscle were measured 24 h after surgery. CLP caused a 50% decrease (from 56 +/- 4 to 29 +/- 2 microM) in plasma ascorbate concentration, 1,000% increase (from 46 +/- 13 to 450 +/- 93 microM) in urine ascorbate concentration, 20% decrease (from 115 +/- 2 to 91 +/- 2 mmHg) in mean arterial pressure, and 30% decrease (from 24 +/- 1 to 17 +/- 1 capillaries/mm) in the density of perfused capillaries, compared with time-matched controls. A bolus of intravenous ascorbate (7.6 mg/100 g body wt) administered immediately after the CLP procedure increased plasma ascorbate concentration and restored both blood pressure and density of perfused capillaries to control levels. In vitro experiments showed that ascorbate (100 microM) inhibited replication of bacteria and prevented hydrogen peroxide injury to cultured microvascular endothelial cells. These results indicate that ascorbate is lost in the urine during sepsis and that a bolus of ascorbate can prevent microvascular dysfunction in the skeletal muscle of septic animals. Our study supports the view that ascorbate may be beneficial for patients with septic syndrome.

Insular cortex stimulation produces lethal cardiac arrhythmias: a mechanism of sudden death?

The rat posterior insular cortex has recently been shown to possess cardiac chronotropic organization and therefore may be involved in cortical mechanisms of sudden death. In order to assess the potential of this region for cardiac arrhythmogenicity, phasic microstimulation of tachycardia zones was undertaken in the urethane-anesthetized rat. The insular stimulus was triggered by the R wave of the electrocardiogram (ECG) and delayed so that resultant putative cardiac sympathetic nerve activity would be synchronous with the T wave of the ECG. This resulted in increasing degrees of heart block leading to escape rhythms, ventricular ectopics and ultimately death in asystole. Heart block was associated with elevated plasma norepinephrine levels and myocardial damage. Such effects have not been previously demonstrated for a cortical site. These data suggest that pathophysiological activation of the insular cortex by stroke, epileptic seizure, or under conditions of severe emotional stress could predispose to ECG changes, cardiac arrhythmias and sudden death.

Cardiac and sympathetic effects of middle cerebral artery occlusion in the spontaneously hypertensive rat.

Acute increases in sympathetic activity, plasma catecholamine concentrations and myocardial damage, occur following middle cerebral artery occlusion (MCAO) in Wistar rats. Hypertension is a major risk factor for stroke. The autonomic responses to MCAO in the spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were therefore investigated. Arterial pressure (AP), heart rate (HR), renal sympathetic nerve discharge (SND), plasma catecholamines and ECG were measured in 16 SHR and 16 WKY male urethane-anesthetized rats, which were subjected to either MCAO or sham MCAO. Cerebral infarct size did not differ between SHR and WKY rats, as shown by tetrazolium staining. Initial AP was significantly higher in SHR (96 +/- 4 mmHg) than in WKY (70 +/- 1 mmHg; P < 0.05). No significant differences in initial HR or plasma catecholamine levels were observed between SHR and WKY. By 6 hours after MCAO, AP, SND and plasma epinephrine in SHR decreased significantly, while HR showed a significant increase. SND and plasma catecholamines in the WKY showed increases that did not reach significant levels following MCAO. The QT interval of the ECG was significantly prolonged in the WKY MCAO rats, which also had a higher frequency of cardiac myocytolysis than the other groups. Unlike the increases in autonomic variables following MCAO in Wistar rats, SHR exhibit significant decreases in SND and AP, while WKY show slight, but non-significant increases. These differences in the autonomic reaction to MCAO may reflect genetic differences in the response to cerebral ischemia.

Plasma and organ catecholamine levels following stimulation of the rat insular cortex.

The posterior insular cortex of the rat contains an area of cardiac chronotropic representation within which tachycardia sites occur rostrally to those producing bradycardia. In the current study using ketamine-anesthetized rats, the insular cortex was stimulated for 1 h using a phasic technique synchronized with the cardiac cycle. Tachycardia was associated with an increase in plasma norepinephrine concentration; epinephrine remained unchanged. This indicates a neural origin of the norepinephrine increment. The tachycardia response was completely blocked by atenolol. Plasma catecholamine levels remained unchanged during stimulation of insular bradycardia sites. Atenolol was without effect during stimulation-induced bradycardia which was completely blocked by atropine. Total cardiac norepinephrine concentration inversely correlated with change in heart rate during stimulation of tachycardia sites. No correlation between intracardiac catecholamines and heart rate variables was found for the bradycardia or control sites. These results indicate that in the ketamine-anesthetized rat, whereas insular stimulation-induced tachycardia is dependent on the sympathetic nervous system, bradycardia elicited by insular cortex stimulation is mediated by parasympathetic mechanisms. No correlation was identified between renal or skeletal muscle norepinephrine levels and any heart rate parameter. This implies that the sympathetic effects of phasic insular microstimulation may be exerted mainly on cardiac nerves, and less so in other visceral beds.

Glutamate stimulates ascorbate transport by astrocytes.

The concentrations of glutamate and ascorbate in brain extracellular fluid increase following seizure activity, trauma and ischemia. Extracellular ascorbate concentration also rises following intracerebral glutamate injection. We hypothesized that glutamate triggers the release of ascorbate from astrocytes. We observed in primary cultures of rat cerebral astrocytes that glutamate increased ascorbate efflux significantly within 30 min. The half-maximal effective concentration of glutamate was 180+/-30 microM. Glutamate-stimulated efflux of ascorbate was attenuated by hypertonic media. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited both Na(+)-dependent glutamate uptake and ascorbate efflux. Two other inhibitors of volume-sensitive organic anion channels (1, 9-dideoxyforskolin and 5-nitro-2-(3-phenylpropylamino) benzoic acid) did not slow glutamate uptake but prevented stimulation of ascorbate efflux. Glutamate also stimulated the uptake of ascorbate by ascorbate-depleted astrocytes. In contrast, glutamate uptake was not affected by intracellular ascorbate, thus ruling out a putative glutamate-ascorbate heteroexchange mechanism. These results are consistent with activation by glutamate of ascorbate-permeant channels in astrocytes.

Sodium-ascorbate cotransport controls intracellular ascorbate concentration in primary astrocyte cultures expressing the SVCT2 transporter.

Expression of the Na(+)-ascorbate cotransporter, SVCT2, was detected in rat brain and in primary cultures of cerebral astrocytes by Northern blot analysis. SVCT2 expression in cultured astrocytes increased in response to the cyclic AMP analog, dibutyryl cyclic AMP. A mathematical model of ascorbic acid transport was developed to evaluate the hypothesis that Na(+)-ascorbate cotransport across the plasma membrane regulates the steady state intracellular concentration of ascorbic acid in these cells. The outcomes predicted by this model were compared to experimental observations obtained with primary cultures of rat cerebral astrocytes exposed to normal and pathologic conditions. Both cotransport activity and intracellular ascorbic acid concentration increased in astrocytes activated by dibutyryl cyclic AMP. Conversely transport activity and ascorbic acid concentration were decreased by hyposmotic cell swelling, low extracellular Na(+) concentration, and depolarizing levels of extracellular K(+). In cells incubated for up to 3 h in medium having an ascorbic acid concentration typical of brain extracellular fluid, the changes in intracellular ascorbic acid concentration actually measured were not significantly different from those predicted by modeling changes in Na(+)-ascorbate cotransport activity. Thus, it was not necessary to specify alterations in vitamin C metabolism or efflux pathways in order to predict the steady state intracellular ascorbic acid concentration. These results establish that SVCT2 regulates intracellular ascorbic acid concentration in primary astrocyte cultures. They further indicate that the intracellular-to-extracellular ratio of ascorbic acid concentration at steady state depends on the electrochemical gradients of Na(+) and ascorbate across the plasma membrane.

Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat.

Stroke models in larger animals such as the cat, dog and monkey are becoming increasingly more expensive and less readily available. However, the rat is an excellent model for focal cerebral ischemia. Rats are readily available, inexpensive and their neuroanatomy and brain function have been studied extensively. Increases in plasma catecholamines and myocardial damage have been observed in clinical stroke. We examined autonomic and myocardial changes in two rat stroke models. In one model only the middle cerebral artery was occluded (MCAO) while the other model involved occlusion of both the MCA and the common carotid artery (MCAO/CCAO). Arterial blood pressure and heart rate were monitored continuously in 25 male rats (326-430 g) that underwent one of the following procedures: (1) MCAO only; (2) MCAO/CCAO; (3) CCAO only; and (4) sham occlusions (SHAM). Arterial blood samples (0.5 ml) for radioenzymatic assay of norepinephrine (NE) and epinephrine (E) were taken twice before the occlusions and at 90 and 180 min after the occlusions. The animals were perfused at the end of the experiment and the heart removed and examined histologically. Tetrazolium salts were reacted with oxidative enzymes to delineate the region of inadequate perfusion. The mean blood pressure and pulse pressure of the SHAM, MCAO/CCAO and CCAO groups significantly declined from initial values (from an average of 78 to 53 mm Hg) during the course of the experiment. However, the mean blood pressure and pulse pressure of the MCAO rats did not change during the experiment, so that the final mean blood pressure and pulse pressure were significantly higher than in the other 3 groups. The levels of both NE and E increased significantly (NE, 1443 +/- 285.9 to 4095 +/- 929 pg/ml; E, 2402 +/- 623 to 3741 +/- 1166 pg/ml) following occlusion in the MCAO group only while the other 3 groups did not change. Four of 6 hearts in the MCAO group were abnormal, showing evidence of subendocardial hemorrhage, ischemic damage or subendocardial congestion. MCAO also resulted in a consistent region of the brain with inadequate perfusion including the insular cortex. These autonomic and myocardial changes appear to mimic some of the changes seen clinically in stroke patients and provide the first acute stroke model for studying autonomic dysfunction in the rat.