Possible cellular mechanism for cerebral vasospasm after experimental subarachnoid hemorrhage in the dog.

This study was undertaken to examine some of the cellular ionic mechanisms responsible for the cerebral vasospasm that occurs as a consequence of subarachnoid hemorrhage (SAH). After cisternal injection of autologous blood we documented spasm of the basilar artery upon angiography from 4 to 7 d postictus in six dogs. When these basilar arteries were isolated we observed marked membrane depolarization and enhanced electrical spike activity compared with controls. The slope of the membrane potential vs log [K]0 curve was significantly reduced in arteries exposed to SAH. Further analysis supported the concept that such altered muscle cell properties resulted from reduction in resting K+ conductance (gk). Exposure of arteries in vitro to nicorandil (10(-9)-10(-7)M) (a drug which acts by increasing gk) hyperpolarized the muscle cells and increased internal diameter. Infusion of nicorandil (3-5 micrograms/kg per min) to intact, anesthetized animals reversed, by 50%, the reduction in basilar artery diameter after experimental SAH.

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca2+ -activated K+ channel current in cultured rat hippocampal astrocytes.

Brief hypoxia differentially regulates the activities of Ca(2+)-activated K(+) channels (K(Ca)) in a variety of cell types. We investigated the effects of hypoxia (<2% O(2)) on K(Ca) channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K(Ca) current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K(Ca) currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K(Ca) channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K(Ca) current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca(2+) external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K(Ca) channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K(Ca) currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K(Ca) single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K(Ca) currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.

Reversal of delayed vasospasm by TS-011 in the dual hemorrhage dog model of subarachnoid hemorrhage.

PURPOSE: Arachidonic acid is avidly metabolized to a potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE), in the cerebral circulation. 20-HETE has been reported to contribute to the acute fall in cerebral blood flow following subarachnoid hemorrhage (SAH), but its role in the development of delayed vasospasm is unknown. The present study examined whether delayed vasospasm is associated with elevations in 20-HETE in CSF in the dual hemorrhage model of SAH in dogs and if blockade of the synthesis of 20-HETE with N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011) can reverse delayed vasospasm in this model. MATERIALS AND METHODS: Delayed vasospasm was induced in 22 adult beagle dogs by dual injection of blood (0.5 mL/kg) into the cisterna magna on days 1 and 4. Sequential samples of CSF were collected before intracisternal injections of blood on days 1 and 4 and after the development of delayed vasospasm on day 7. Sequential angiograms were obtained before and after intracisternal injection of blood on days 1 and 4 and before and 1 hour after administration of TS-011 (1 mg/kg IV) on day 7. RESULTS: The dogs consistently developed delayed vasospasm, and the diameter of the basilar artery fell to 68 +/- 3% (n = 15), 3 days after the second intracisternal injection of blood. The levels of 20-HETE in CSF increased from 4 +/- 2 to 39 +/- 16 pg/mL. In 9 dogs with delayed vasospasm, acute blockade of the synthesis of 20-HETE with TS011 (1 mg/kg IV) significantly increased the diameter of the basilar artery by 39%. Chronic administration of TS-011 (1 mg/kg per day) attenuated the development of delayed vasospasm, and the diameter of the basilar artery fell by 17 +/- 1% versus the 33 +/- 3% decrease in diameter seen in control animals 3 days following the second injection of blood into the cisterna magna. CONCLUSIONS: These results indicate that the development of delayed vasospasm in dogs is associated with an increase in 20-HETE levels in CSF, and acute blockade of the synthesis of 20-HETE with TS-011 reverses delayed vasospasm in this model.

Transduction of physical force by the vascular wall Role of phospholipase C and cytochrome P450 metabolites of arachidonic acid.

The blood vessel wall responds actively to an elevation in transmural pressure. This pressure-induced myogenic response is thought to set the basal level of vascular tone upon which metabolic and neural influences operate in concert to regulate organ blood flow. The cellular mechanisms that mediate the vascular muscle response to mechanical deformation via a changing transmural pressure include membrane depolarization, activation of phospholipase C, and a rise in intracellular [Ca(2+)](i), which appear to be nonadapting-remaining active as long as the pressure stimulus is applied. This brief review addresses some of the cellular events mediating transduction of transmural pressure by the vessel wall. Two possible mechanisms that are responsible for the nonadapting nature of pressure-induced myogenic tone are also explored, namely, formation of a P450 metabolite of arachidonic acid, which acts to buffer activation of K(+) channels as intracellular Ca(2+) rises, and direct activation of Ca(2+) channels by diacylglycerol. Evidence is provided suggesting that activation of phospholipase C is responsible for both the release of the arachidonic acid substrate for P450 enzymes and for the formation of diacylglycerol via its action on membrane-bound phospholipids.

Membrane mechanisms in arterial hypertension.

The purpose of this review is to focus on alterations in vascular muscle membrane potentials (Em), ionic permeabilities, and ionic transport systems which may either contribute to or be a consequence of the hypertensive state. Three models of hypertension are discussed: 1) deoxycorticosterone-salt (DOCA-salt)-induced hypertension; 2) low-renin (presumably volume expanded) renal hypertension (LRRH); and 3) the spontaneously hypertensive rat (SHR) of the Okamoto-Aoki Kyoto-Wistar strain and its normotensive genetic control (WKY). The importance of studying all possible mechanisms of increased contraction in vascular smooth muscle is stressed.

Enhanced vascular tone in the renal vasculature of spontaneously hypertensive rats.

The renal microvascular responses of Wistar-Kyoto and spontaneously hypertensive rats to changes in perfusion pressure were compared using a juxtamedullary nephron microvascular preparation perfused in vitro with a physiological salt solution containing 5% albumin. In the spontaneously hypertensive rats, the internal diameters of arcuate and interlobular arteries and the proximal and distal afferent arterioles averaged 307 +/- 26, 52 +/- 2, 24 +/- 0.9, and 22 +/- 1.2 microns, respectively, at 80 mm Hg. They were 18-35% smaller (p less than 0.05) than the corresponding vessels measured in Wistar-Kyoto rats. In low calcium media, the arcuate and interlobular arteries and the proximal and distal afferent arterioles of spontaneously hypertensive rats exhibited a greater dilation than the vessels of Wistar-Kyoto rats. These observations suggest that the diameters of the preglomerular vasculature of the spontaneously hypertensive rats are reduced because of an elevated vascular tone rather than structural changes narrowing the lumen of these vessels. These results suggest that enhanced vascular tone in the preglomerular vasculature of juxtamedullary nephrons may contribute to the elevated renal medullary vascular resistance and resetting of the pressure-natriuretic relation previously observed in spontaneously hypertensive rats.

Elevated renovascular tone in young spontaneously hypertensive rats. Role of cytochrome P-450.

The present study examined the role of cytochrome P-450 metabolites of arachidonic acid in elevating renal vascular resistance in young spontaneously hypertensive rats (SHR). Differences in vascular tone were assessed in the preglomerular vasculature of 3- to 4-week-old prehypertensive SHR (n = 11) and normotensive Wistar-Kyoto (WKY, n = 10) and Wistar-Lewis (n = 10) rats. Pressure-diameter relations to changes in renal perfusion pressure were compared using the juxtamedullary nephron microvascular preparation perfused in vitro with a physiological salt solution. At a pressure of 60 mm Hg, the basal diameters of the interlobular arteries and proximal and distal afferent arterioles of the SHR averaged 43 +/- 2, 17 +/- 0.3, and 11 +/- 0.4 microns, respectively. The diameters of the interlobular arteries and afferent arterioles were 9% to 14% smaller than those of corresponding vessels in WKY and Wistar-Lewis rats. Addition of P-450 inhibitors, ketoconazole (100 mumol/L) or 7-ethoxyresorufin (1 mumol/L), to the perfusate dilated the afferent arteriole of SHR by 7% to 12%, whereas it increased the diameter by only 0% to 6% in control rats and significantly reduced the differences in the pressure-diameter relation in the preglomerular vasculature of SHR and control rats. Inhibitors of P-450 eliminated the contractile response of afferent arterioles to increases in renal perfusion pressure in all three groups. Removal of calcium from the perfusate eliminated differences in the diameters of the preglomerular vasculature in SHR and normotensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanine nucleotide-binding proteins in aortic smooth muscle from hypertensive rats.

To explore the hypothesis that altered vascular muscle signal transduction may underlie some of the vascular changes observed in hypertensive models, we measured expression of GTP-binding protein (G protein) alpha-subunits, Gs, G(i), and Gq, in aortic muscle of reduced renal mass and sham-operated rats and proximal and distal aortic segments from rats with interrenal aortic coarctation (IR-AC). G protein expression was measured by immunoblot analysis. When we probed aortic muscle membrane with G(i) and Gq alpha-subunit antibodies, we identified 41- and 42-kD immunoreactive proteins, respectively. Three immunoreactive bands specific to Gs alpha-subunit antibody were resolved. Immunoreactive blot densities were compared. In aortic muscle membrane of reduced renal mass rats (blood pressure, 148 +/- 7 mm Hg), we found significantly reduced Gs and G(i) blot densities compared with sham-operated controls (blood pressure, 99 +/- 12 mm Hg). There were no differences in Gq blot densities between reduced renal mass and control rats. Gs and G(i) blot densities were significantly lower in IR-AC proximal aortic segments (carotid pressure, 165 +/- 5 mm Hg) and distal aortic segments (femoral pressure, 121 +/- 4 mm Hg) than in aortas of sham-operated controls. In contrast, Gq expression was significantly increased in the high-pressure proximal aortic segments compared with low-pressure distal aortic segments from IR-AC rats. Thus, altered G protein expression occurs in aortic muscle from nongenetic rat models of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of tyrosine kinase and PKC in the vasoconstrictor response to 20-HETE in renal arterioles.

The present study examined the hypothesis that activation of protein kinase C (PKC), components of the mitogen-activated protein (MAP) kinase pathway, or both contributes to the inhibitory effects of 20-hydroxyeicosatetraenoic acid (20-HETE) on K+-channel activity and its vasoconstrictor response in renal arterioles. 20-HETE (0.1 to 50 micromol/L) dose-dependently produced a 30% increase in PKC activity and a fivefold rise in the expression of active extracellular signal-regulated kinase 1 (ERK1) and ERK2 proteins in renal microvessels. 20-HETE (0.01 to 1 micromol/L) reduced the diameter of isolated perfused renal interlobular arterioles by 33+/-2%. Blockade of PKC activity with an N-myristoylated PKC pseudosubstrate inhibitor (Myr-PKCi, 100 micromol/L) or calphostin C (0.5 micromol/L) had no significant effect on the vasoconstrictor response to 20-HETE. In contrast, the tyrosine kinase inhibitors genistein (30 micromol/L) and tyrphostin 25 (10 micromol/L) reduced the response to 20-HETE by 76.5+/-2.1% and 67.5+/-1.8%, respectively. A specific inhibitor of mitogen-activated extracellular signal-regulated kinase (MEK), PD98059, had no effect on the vasoconstrictor response to 20-HETE. In cell-attached patches on renal vascular smooth muscle cells, 20-HETE reduced the open state probability of a large-conductance K+ channel (from 0.0026+/-0.0004 to 0.0006+/-0.0001). The Myr-PKCi (100 micromol/L) did not alter the inhibitory effects of 20-HETE on this channel. In contrast, the tyrosine kinase inhibitor genistein (30 micromol/L) blocked the inhibitory effects of 20-HETE on the large-conductance K+ channel. These data suggest that 20-HETE activates the MAP kinase system in renal arterioles and that the activation of a tyrosine kinase, which is proximal to MEK in this cascade, contributes to the inhibitory effects of 20-HETE on K+-channel activity and its vasoconstrictor effects in the renal arterioles.

Spontaneous flow oscillations in the cerebral cortex during acute changes in mean arterial pressure.

The purpose of this study was to characterize spontaneous oscillations of blood flow in the cerebral cortex of anesthetized rats under control conditions and after mean arterial pressure was altered by various means. Blood flow was monitored using a laser-Doppler flowmeter through the closed cranium. Spontaneous flow oscillations with amplitudes of 14-30% of the mean flow and frequencies of 4-11 cycles/min were recorded when arterial pressures were less than 90 mm Hg. Stepwise hemorrhagic hypotension and unilateral carotid occlusion increased the amplitude of oscillations. The amplitude of oscillations was negatively correlated with the level of mean arterial pressure after manipulation with norepinephrine or sodium nitroprusside. The oscillations were reversibly abolished during dilation of the cerebral circulation by elevating the inspired carbon dioxide content to 5%. The frequency of flow oscillations was very stable during all of the above maneuvers except during the infusion of norepinephrine, which increased the oscillation frequency slightly. The results suggest that flow oscillations are determined primarily by cerebral arterial pressure.

A membrane electrical mechanism for hypoxic vasoconstriction of small pulmonary arteries from cat.

Using specially fabricated muscle myographs, we examined electrical and mechanical responses to reduction of PO2 in small (less than 300 micron) pulmonary arteries excised from cat lungs. Upon lowering PO2 from 400 to 50 mm Hg, these preparations consistently developed contractile responses concomitant with membrane depolarization and action potential generation. The largest changes in electromechanical responses to reduction of PO2 occurred between 150 and 50 mm Hg. These data strongly suggest that hypoxic activation of small pulmonary arteries is mediated by direct effects of reduced PO2 on muscle cell membrane ionic conductance systems.

Hypoxic induction of Ca2+-dependent action potentials in small pulmonary arteries of the cat.

Small pulmonary arteries (less than 300 micron) from cats were mounted in myographs to record mechanical and electrical responses to hypoxia. When these preparations were exposed to a PO2 of 30-50 Torr after equilibration at 300 Torr they consistently developed active force, which increased or decreased in amplitude as [Ca2+] was raised or lowered, respectively, and was blocked on addition of verapamil. Intracellular electrical recording with glass microelectrodes demonstrated membrane depolarization and action potential generation when PO2 was lowered. Steady-state voltage vs. applied current curves obtained before and during hypoxia showed a significant reduction in input resistance. The relationship between membrane potential and extracellular K+ was not different during hypoxia compared with control, suggesting that there were not marked changes in K+ permeability under this condition. In the presence of verapamil to block Ca2+ inward current the hypoxia-induced action potentials were abolished concomitant with partial membrane repolarization. The results of these studies suggest that in certain isolated pulmonary arteries hypoxia induces contraction by a mechanism involving an increased Ca2+ conductance. These data suggest that the sensor involved in hypoxic pulmonary vasoconstriction may lie within the vessel wall and somehow mediates changes in smooth muscle ionic conductances.

Hypoxia-induced activation in small isolated pulmonary arteries from the cat.

Effects of hypoxia on force development and membrane potential were studied in isolated small (less than 300 microns diam) and large (greater than 500 microns diam) pulmonary arteries from cats. There was a consistent and reproducible hypoxic constrictor response in small pulmonary arteries that began at PO2 values between 350 and 300 Torr and reached a maximum at PO2 between 50 and 30 Torr. In the small artery smooth muscle cell the membrane potential, which was -51 +/- 1.4 mV at a PO2 of 400 Torr, was depolarized to -37 +/- 2 mV at a PO2 of 50 Torr. In contrast, larger arteries did not exhibit significant hypoxic constriction or depolarization upon exposure to low PO2. Constriction in small arteries was not blocked by phentolamine. Treatment with a low dose of indomethacin (10(-9) M) augmented the response; however, a larger dose of indomethacin (10(-3) M) blocked the constriction to hypoxia but not to 30 mM KCl. Depolarization during hypoxia was not blocked by ouabain. Results of this study suggest that the hypoxic response of these isolated small pulmonary vessels may be like that seen in the intact lung. Furthermore, these data suggest that hypoxic vasoconstriction may be mediated by electrical events occurring at the pulmonary arterial muscle cell membrane either directly or via mediators released from the vessel wall.

Distensibility of small arteries of the dog lung.

To obtain in situ measurements of the distensibility of small (100- to 1,000-microns-diam) pulmonary arterial vessels of the dog lung, X-ray angiograms were obtained from isolated lung lobes with the vascular pressure adjusted to various levels. The in situ diameter-pressure relationships were compared with the diameter-pressure relationships for small arteries that were dissected free from the lungs and cannulated with small glass pipettes for the measurement of diameter and transmural pressure. The diameter-vascular or diameter-transmural pressure curves from both in situ and cannulated vessels were sufficiently linear in the pressure range studied (0-30 Torr) that they could be characterized by linear regression to obtain estimates of D0, the diameter at zero vascular pressure, and beta, the change in diameter (micron) per Torr change in pressure. The vessel distensibility coefficient (alpha) was defined as alpha = beta/D0. The mean values of alpha were approximately 2.0 +/- 0.8%/Torr (SD) for the in situ vessels and 1.7 +/- 0.6%/Torr for the cannulated vessels, with no statistically significant difference between the two methods. The influence of vasoconstriction elicited by serotonin was evaluated in the in situ vessels. Serotonin-induced vasoconstriction caused a decrease in D0 and little change in alpha.

Distensibility of small veins of the dog lung.

To determine the distensibility of the intrapulmonary veins (250-2,900 microns diam) of the dog lung, we obtained X-ray angiograms from isolated lung lobes over a vascular pressure range of approximately 0-30 Torr. Over this pressure range the diameter vs. pressure curves tended to flatten out at the high pressures. In the pressure range of 0-19 Torr, we characterized the vessel distensibility by alpha (the ratio of the slope, beta, of the graph of diameter vs. intravascular pressure to the intercept, Do). The average value of alpha was approximately 1.2%/Torr. There was a weak negative correlation (r = -0.32) between alpha and Do. Infusion of enough norepinephrine to produce approximately 50% increase in total lobar vascular resistance produced a decrease in Do and alpha of approximately 33 and 32%, respectively.

Electromechanical coupling in feline basilar artery in response to serotonin.

This study was undertaken to define some of the cellular mechanisms of action of serotonin on cerebral vascular muscle. Application of serotonin to cat basilar artery resulted in a dose dependent depolarization and contraction beginning at 10(-8) M. The correlation coefficient relating the degree of force development with the change in membrane potential (Em) was 0.98. Excess K+ depolarized these vascular muscle cells with a slope (between 10 and 100 mM [K]0) of 54 mV/decade. When the muscle cells within this artery were depolarized by only 7 mV by addition of excess K+ there was a significant reduction in force development in response to serotonin. When the membrane was depolarized from -63 to -40 mV the mechanical response to serotonin was reduced by around 50%. Steady state current/voltage curves demonstrated a reduction in input resistance suggesting that serotonin's mechanism of depolarization is not due to a reduction in gk. These data demonstrate that serotonin contracts cat basilar artery through mechanisms involving vascular muscle cell depolarization and that factors which influence the level of Em will markedly effect the contractile response to serotonin.

Cellular mechanisms of opiate receptor stimulation in cat middle cerebral artery.

To determine some of the cellular mechanisms of opiate receptor stimulation in cat middle cerebral arterial muscle, intracellular electrical measurements and force development were monitored before and after addition of morphine. Addition of morphine resulted in a dose-dependent hyperpolarization of the muscle cells in the middle cerebral artery with a concomitant relaxation, indicating a high degree of electromechanical coupling in this preparation. The curve relating membrane potential vs. morphine was shifted to the right and downward by naloxone, demonstrating competitive inhibition at receptor sites. When middle cerebral arteries were studied from animals which had been injected with morphine prior to sacrifice, a significant hyperpolarization of the membrane was recorded when studied in an organ bath. This hyperpolarization was abolished if the animal had been pretreated with naloxone prior to morphine injection, suggesting that morphine may act in vivo as we have observed it to act in vitro. Morphine-induced hyperpolarization could be blocked in the organ bath when potassium conductance (gk) was inhibited. Similarly, the reduction in the slope of the voltage/current curve induced by morphine was blocked by agents which reduced gk. These data suggest the presence of opiate receptors on cat cerebral artery and suggest that morphine relaxes these vessels through a mechanism involving increased gk. These findings suggest a role for opiate-mediated systems in cerebral vascular control.

Early effects of experimental diabetes on central catecholamine concentrations.

Six days after induction of diabetes norepinephrine (NE), 3,4-dihydroxyphenylalanine and dopamine concentrations were determined in spinal cord, cerebellum, pons/medulla oblongata and the remaining brain of diabetic rats. Only cerebellar NE levels were significantly increased by 21% compared to age-matched controls. This effect was not seen when diabetic rats received insulin replacement therapy. Fifty-two days after induction of diabetes cerebellar NE levels in diabetic rats were further increased (+37%), implying an enduring and progressive effect. The data show that even with a short period of uncontrolled diabetes central neurochemical alterations occur.

Electromechanical coupling in rat basilar artery in response to morphine.

Force development, intracellular membrane potential (Em), and voltage vs. current curves were measured in rat basilar artery to help elucidate the mechanism of action of morphine sulfate and a synthetic narcotic, meperidine hydrochloride, on this preparation. Morphine sulfate caused a dose-dependent contraction of these vessels, which was reversible with naloxone. Electrical studies show that morphine may act upon this vascular smooth muscle preparation by decreasing potassium conductance (gk). This hypothesis is supported by the findings that morphine sulfate depolarized these cells and increased the input resistance (rin) determined by the application of rectangular hyperpolarizing and depolarizing current pulses through the microelectrode during impalement and recording of the associated voltage changes (delta V). Meperidine hydrochloride had significantly less effect on this preparation than morphine sulfate. Further studies show that the vehicular medium used for the commercially available preparation of naloxone (viz. the methyl and propyl esters of p-hydroxybenzoic acid in a ratio of 9:1) is, in vitro, a vasodilator of cerebral vascular smooth muscle.

Altered splenic catecholamine concentrations during experimental allergic encephalomyelitis.

Catecholamine concentrations of the spleen were studied with neurochemical techniques in rats injected with myelin basic protein to produce an experimental allergic encephalomyelitis (EAE). Thirteen to 14 days postinoculation the affected rats showed peak clinical signs of weakness, especially in the lower extremities. Resolution of the disease then progressed rapidly with full clinical recovery at day 21. Splenic concentrations of norepinephrine (NE), 3,4-dihydroxyphenylalanine (DOPA), epinephrine (EPI) and 3,4-dihyxroxyphenylethylamine (DA) were determined by HPLC with electrochemical detection. DOPA concentrations were significantly increased (+62%) while DA concentrations were decreased (-29%) in the EAE rats on day 14 postinoculation. NE and EPI concentrations tended to be elevated in the EAE group, but this was not statistically significant. No differences in splenic catecholamines were detected on day 7 and 52 postinoculation between EAE and control animals. These results indicate that changes in the metabolic pathways of splenic catecholamines occur at the peak of the clinical symptoms of EAE; the increase in DOPA and the decrease in DA concentrations suggest that the activity of DOPA-decarboxylase or its co-factor is altered.