Cocaine Constrictor Mechanisms of the Cerebral Vasculature.

Cocaine constriction of the cerebral vasculature is thought to contribute to the ischemia associated with cocaine use. However, the mechanisms whereby cocaine elicits relevant vasoconstriction remain elusive. Indeed, proposed intra- and intercellular mechanisms based on over 3 decades of ex vivo vascular studies are, for the most part, of questionable relevancy due to the generally low contractile efficacy of cocaine combined with the use of nonresistance-type vessels. Furthermore, the significance attached to mechanisms derived from in vivo animal studies may be limited by the inability to demonstrate cocaine-induced decreased cerebral blood flow, as observed in (awake) humans. Despite these apparent limitations, we surmise that the vasoconstriction relevant to cocaine-induced ischemia is elicited by inhibition of dilator and activation of constrictor pathways because of cocaine action on the neurovascular unit (neuron, astrocyte, and vessel) and on vessels outside the unit. Furthermore, previous cocaine exposure, that is, conditions present in human subjects, downregulates and sensitizes these dilator and constrictor pathways, respectively, thereby enhancing constriction to acute cocaine. Identification of specific intra- and intercellular mechanisms requires investigations in the isolated microvasculature and the neurovascular unit from species chronically exposed to cocaine and in which cocaine decreases cerebral blood flow.

Functional ET(A)-ET(B) Receptor Cross-talk in Basilar Artery In Situ From ET(B) Receptor Deficient Rats.

The role of endothelin (ET)(A)-ET(B) receptor cross-talk in limiting the ET(A) receptor antagonist inhibition of ET-1 constriction is revealed by the partial or complete dependency of the ET(A) receptor antagonist inhibition on functional removal of the ET(B) receptor. Although functional removal of the ET(B) receptor is generally accomplished with ET(B) receptor antagonist, a novel approach using rats containing a naturally occurring deletion mutation in the ET(B) receptor [rescued "spotting lethal" (sl) rats; ET(B)(sl/sl)] demonstrated increased ET(A) receptor antagonist inhibition of ET-1 constriction in vena cava. We investigated whether this deletion mutation was also sufficient to remove the ET(B) receptor dependency of the ET(A) receptor antagonist inhibition of ET-1 constriction in the basilar artery. Consistent with previous reports, ET-1 plasma levels were elevated in ET(B)(sl/sl) as compared with ET(B)(+/+) rats. ET(B) receptor antagonist failed to relax the ET-1 constricted basilar artery from ET(B)(+/+) and ET(B)(sl/sl) rats. Relaxation to combined ET(A) and ET(B) receptor antagonist was greater than relaxation to ET(A) receptor antagonist in the basilar artery from ET(B)(+/+) and, unexpectedly, ET(B)(sl/sl) rats. These findings confirm the presence of ET(A)-ET(B) receptor cross-talk in the basilar artery. We speculate that mutant ET(B) receptor expression produced by alternative splicing may be sufficient to allow cross-talk.

Cocaine constriction of rat basilar artery in situ: roles of nitric oxide and endothelin-1.

This study investigated whether cocaine constriction of rat basilar artery in situ is mediated by nitric oxide (NO) inhibition and/or endothelin (ET)-1 release. Cocaine (3-100 µmol/l) concentration-dependently constricted the basilar artery to a maximum of 18%. Nω-nitro-L-arginine (100 µmol/l) was without effect on constriction to 3 and 10 µmol/l cocaine. PD145065 (1 and 10 µmol/l), an ETA/B receptor antagonist, variably and at most partially inhibited the 100 µmol/l cocaine constriction. Capsaicin denervation of sensory nerves innervating the basilar, which contain ET-1 and NO synthase, also failed to influence cocaine constriction. These findings suggest that cocaine constriction of cerebral vessels (1) varies with respect to the involvement of ET-1 release and (2) unlike findings in the peripheral vasculature, the constriction is not mediated by inhibition of NO.

Endothelin(A)-endothelin(B) receptor cross talk in endothelin-1-induced contraction of smooth muscle.

The efficacy of selective endothelin (ET) receptor antagonists may be limited by a functional interaction between the ET(A) and ET(B) receptors. This interaction, also termed "cross talk", is characterized by the dependency of the inhibition of an ET-1 response due to antagonism of one ET receptor subtype upon concomitant antagonism of the other ET receptor subtype. Although a reduction in ET(A)-ET(B) receptor cross talk would presumably increase the efficacy of selective ET receptor antagonists, an approach that accomplishes this aim is largely absent due to a lack of mechanistic understanding. Toward this goal, we evaluated the characteristics and potential dependencies of cross talk in smooth muscle. Smooth muscle was adopted as an exemplar not only because cross talk is widely reported in this tissue type, thereby allowing numerous comparisons, but also significant controversy surrounds the use of selective versus nonselective ET receptor antagonists in ET-1-related pathophysiologies involving smooth muscle. Based on this evaluation, we suggest that ET(A)-ET(B) receptor cross talk is a dynamic process directed by either or both ET receptor subtypes and expressed to varying magnitudes depending on the ET-1 and selective ET receptor antagonist concentrations, tone due to intraluminal pressure/stretch, agonists acting at receptors other than the ET(A)/ET(B) receptors, and endothelial/epithelial function. It is speculated that ET(A)-ET(B) receptor cross talk occurs through signal transduction pathways along with changes at the receptor level. Pharmacologic intervention of the signaling pathways could increase the therapeutic efficacy of ET receptor antagonists.

Mechanism of Thiazide Diuretic Arterial Pressure Reduction: The Search Continues.

Thiazide diuretic (TZD)-mediated chronic reduction of arterial pressure is thought to occur through decreased total peripheral vascular resistance. Further, the decreased peripheral vascular resistance is accomplished through TZD activation of an extrarenal target, resulting in inhibition of vascular constriction. However, despite greater than five decades of investigation, little progress has been made into the identification of the TZD extrarenal target. Proposed mechanisms range from direct inhibition of constrictor and activation of relaxant signaling pathways in the vascular smooth muscle to indirect inhibition through decreased neurogenic and hormonal regulatory pathways. Surprisingly, particularly in view of this lack of progress, comprehensive reviews of the subject are absent. Moreover, even though it is well recognized that 1) several types of hypertension are insensitive to TZD reduction of arterial pressure and, further, TZD fail to reduce arterial pressure in normotensive subjects and animals, and 2) different mechanisms underlie acute and chronic TZD, findings derived from these models and parameters remain largely undifferentiated. This review 1) comprehensively describes findings associated with TZD reduction of arterial pressure; 2) differentiates between observations in TZD-sensitive and TZD-insensitive hypertension, normotensive subjects/animals, and acute and chronic effects of TZD; 3) critically evaluates proposed TZD extrarenal targets; 4) proposes guiding parameters for relevant investigations into extrarenal TZD target identification; and 5) proposes a working model for TZD chronic reduction of arterial pressure through vascular dilation.

Lack of thiazide diuretic inhibition of agonist constriction of mouse mesenteric arterioles ex vivo.

The chronic reduction of arterial blood pressure by thiazide diuretics (TZD) in hypertensive patients is mediated through an extra-renal mechanism. It is widely held that this extra-renal mechanism is a direct TZD inhibition of vasoconstriction. This study tested whether the TZD, hydrochlorothiazide (HCTZ), inhibited agonist constriction of mesenteric arterioles ex vivo. Mice deficient in the kidney distal convoluted tubule Na+/Cl- cotransporter (NCC), i.e., the target of thiazide inhibition-mediated diuresis, and wild type (WT), were subjected to Na+-restricted diet. Mesenteric arterioles from NCC knockout and WT mice were then isolated, placed under constant pressure, and the inhibitory effects of HCTZ (100 µM) on phenylephrine constriction determined. HCTZ did not inhibit phenylephrine constriction of arterioles from NCC knockout and wild type (WT) mice subjected to Na+-restricted diet. This study suggests that future investigations to identify the extra-renal site of chronic TZD treatment should (1) focus on indirect inhibition of vascular constriction and (2) be determined under clinically relevant conditions. These conditions include chronic TZD at relevant concentrations in hypertensive animals.

Bilirubin Oxidation Products and Cerebral Vasoconstriction.

Key evidence in support of the hypothesis that bilirubin oxidation products (BOXes) contribute to the vasoconstriction associated with subarachnoid hemorrhage (SAH) are the (1) presence of BOXes in cerebral spinal fluid from SAH patients and (2) ability of one or more BOXes to elicit vasoconstriction. We critically evaluate this key evidence, detail where gaps remain, and describe recent approaches that will address these gaps.

UV light absorption parameters of the pathobiologically implicated bilirubin oxidation products, MVM, BOX A, and BOX B.

The formation of the bilirubin oxidation products (BOXes), BOX A ([4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide]) and BOX B (3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide), as well as MVM (4-methyl-3-vinylmaleimide) were synthesized by oxidation of bilirubin with H2O2 without and with FeCl3, respectively. Compound identity was confirmed with NMR and mass spectrometry (MS; less than 1 ppm, tandem MS up to MS4). UV absorption profiles, including λmax, and extinction coefficient (ε; estimated using NMR) for BOX A, BOX B, and MVM in H2O, 15% CH3CN plus 10 mM CF3CO2H, CH3CN, CHCl3, CH2Cl2, and 0.9% NaCl were determined. At longer wavelengths, λmax's for 1) BOX A were little affected by the solvent, ranging from 295-297 nm; 2) BOX B, less polar solvent yielded λmax's of lower wavelength, with values ranging from 308-313 nm, and 3) MVM, less polar solvent yielded λmax's of higher wavelength, with values ranging from 318-327 nm. Estimated ε's for BOX A and BOX B were approximately 5- to 10-fold greater than for MVM.

Acute cocaine induces endothelin-1-dependent constriction of rabbit basilar artery.

It has been postulated that ischemic stroke due to acute cocaine usage involves constriction of the cerebral vasculature. However, the mechanism underlying the constriction remains unclear. This study tested whether cocaine constriction was mediated via endothelin-1. Cocaine suffusion induced maintained constriction in the rabbit basilar artery in situ. The constriction was relaxed by PD145065, an endothelin A and B receptor antagonist. These results support the hypothesis that constriction of the cerebral vasculature due to acute cocaine exposure is via endothelin-1 release. Endothelin receptor antagonists may be of therapeutic benefit in cerebrovascular pathophysiologies involving cocaine constriction.

Vascular contractile reactivity in hypotension due to reduced renal reabsorption of Na+ and restricted dietary Na.

Reduced renal Na+ reabsorption along with restricted dietary Na+ depletes intravascular plasma volume which can then result in hypotension. Whether hypotension occurs and the magnitude of hypotension depends in part on compensatory angiotensin II-mediated increased vascular resistance. We investigated whether the ability of vascular resistance to mitigate the hypotension was compromised by decreased contractile reactivity. In vitro reactivity was investigated in aorta from mouse models of reduced renal Na+ reabsorption and restricted dietary Na+ associated with considerable hypotension and renin-angiotensin system activation: (1) the Na+-Cl--Co-transporter (NCC) knockout (KO) with Na+ restricted diet (0.1%, 2 weeks) and (2) the relatively more severe pendrin (apical chloride/bicarbonate exchanger) and NCC double KO. Contractile sensitivity to KCl, phenylephrine, and/or U46619 remained unaltered in aorta from both models. Maximal KCl and phenylephrine contraction expressed as force/aorta length from NCC KO with Na+-restricted diet remained unaltered, while in pendrin/NCC double KO were reduced to 49 and 64%, respectively. Wet weight of aorta from NCC KO with Na+-restricted diet remained unaltered, while pendrin/NCC double KO was reduced to 67%, consistent with decreased medial width determined with Verhoeff-Van Gieson stain. These findings suggest that hypotension associated with severe intravascular volume depletion, as the result of decreased renal Na+ reabsorption, may in part be due to decreased contractile reactivity as a consequence of reduced vascular hypertrophy.

Endothelin-1 Regulation of Exercise-Induced Changes in Flow: Dynamic Regulation of Vascular Tone.

Although endothelin (ET)-1 is a highly potent vasoconstrictor with considerable efficacy in numerous vascular beds, the role of endogenous ET-1 in the regulation of vascular tone remains unclear. The perspective that ET-1 plays little role in the on-going regulation of vascular tone at least under physiologic conditions is supported by findings that potential ET-1 constriction is minimized by the release of the vasodilator and ET-1 synthesis inhibitor, nitric oxide (NO). Indeed, ET-1 release and constriction is self-limited by ET-1-induced, endothelial ETB receptor-mediated release of NO. Moreover, even if the balance between ET-1 and NO were reversed as the result of lowered NO activity, as occurs in a number of pathophysiologies associated with endothelial dysfunction, the well-known resistance of ET-1 constriction to reversal (as determined with exogenous ET-1) precludes ET-1 in the dynamic, i.e., moment-to-moment, regulation of vascular tone. On the other hand, and as presently reviewed, findings of ET-1-dependent modulation of organ blood flow with exercise under physiologic conditions demonstrate the dynamic regulation of vascular tone by ET-1. We speculate that this regulation is mediated at least in part through changes in ET-1 synthesis/release caused by pulsatile flow-induced shear stress and NO.

The non-diuretic hypotensive effects of thiazides are enhanced during volume depletion states.

Thiazide derivatives including Hydrochlorothiazide (HCTZ) represent the most common treatment of mild to moderate hypertension. Thiazides initially enhance diuresis via inhibition of the kidney Na+-Cl- Cotransporter (NCC). However, chronic volume depletion and diuresis are minimal while lowered blood pressure (BP) is maintained on thiazides. Thus, a vasodilator action of thiazides is proposed, likely via Ca2+-activated K+ (BK) channels in vascular smooth muscles. This study ascertains the role of volume depletion induced by salt restriction or salt wasting in NCC KO mice on the non-diuretic hypotensive action of HCTZ. HCTZ (20mg/kg s.c.) lowered BP in 1) NCC KO on a salt restricted diet but not with normal diet; 2) in volume depleted but not in volume resuscitated pendrin/NCC dKO mice; the BP reduction occurs without any enhancement in salt excretion or reduction in cardiac output. HCTZ still lowered BP following treatment of NCC KO on salt restricted diet with paxilline (8 mg/kg, i.p.), a BK channel blocker, and in BK KO and BK/NCC dKO mice on salt restricted diet. In aortic rings from NCC KO mice on normal and low salt diet, HCTZ did not alter and minimally decreased maximal phenylephrine contraction, respectively, while contractile sensitivity remained unchanged. These results demonstrate 1) the non-diuretic hypotensive effects of thiazides are augmented with volume depletion and 2) that the BP reduction is likely the result of HCTZ inhibition of vasoconstriction through a pathway dependent on factors present in vivo, is unrelated to BK channel activation, and involves processes associated with intravascular volume depletion.

Alkaline pH-induced extracellular regulated protein kinase activation in brain microvascular endothelial cells: differential effects of Tris and lowered CO2.

As it was previously reported that Tris-elevated pH acutely activated extracellular regulated protein kinase (ERK) in rat aorta smooth muscle cells, this study tested whether this finding could be extended to endothelial cells and, moreover, the relevance of this finding in brain microvascular endothelial cells with respect to respiratory-induced hypocapnic alkalosis. Exposure of bovine brain microvascular endothelial cells to pH 7.90 due to Tris for 15 and 30 min activated ERK twofold. In contrast, pH elevated to 7.75 and 7.90 by lowered percent CO2 failed to activate ERK (15, 30, and 60 min). These results suggest that respiratory alkalosis due to hypocapnia does not activate ERK in brain microvascular endothelial cells. The ability of Tris to activate ERK suggests a novel pathway, possibly independent of pH elevation, whereby Tris activates ERK.

Vasospasm following subarachnoid hemorrhage: evidence against functional upregulation of protein kinase C constrictor pathway.

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of protein kinase C. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Chelerythrine, a protein kinase C inhibitor, relaxed the spasm. However, relaxations to chelerythrine were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that subarachnoid hemorrhage induced vasospasm does not involve functional upregulation of protein kinase C.

Vasospasm following subarachnoid hemorrhage: evidence against functional upregulation of Rho kinase constrictor pathway.

This study tested the hypothesis that vasospasm due to subarachnoid hemorrhage involves the functional upregulation of Rho kinase. Spasm of the rabbit basilar artery was achieved using a double hemorrhage model, which we previously demonstrated was endothelin-1 dependent. In situ effects of agents were determined by direct measurement of vessel diameter following their suffusion in a cranial window. Y-27632, a Rho kinase inhibitor, relaxed the spasm. However, relaxations to Y-27632 were not significantly greater in endothelin-1 constricted spastic vessels initially relaxed with the endothelin converting enzyme inhibitor, phosphoramidon, as compared to endothelin-1 constricted control vessels. These results suggest that, at least in the rabbit double subarachnoid hemorrhage model, vasospasm does not involve the functional upregulation of Rho kinase.

Repeated constriction to respiration-induced hypocapnia is not mimicked by isocapnic alkaline solution in rabbit basilar artery in situ.

The purpose of this study was to test whether constriction of the cerebral vasculature in response to respiration-induced hypocapnia was mimicked by isocapnic alkaline solution. Since the regulation of the cerebral vasculature by hypocapnia necessitates vessels to constrict repeatedly in response to hypocapnic challenge, we tested whether repeated challenge with isocapnic alkaline solution was also associated with constriction. In contrast to our previous demonstration that repeated hypocapnic challenge elicited constrictions of similar magnitudes in rabbit basilar artery in situ, repeated challenge with isocapnic alkaline solution resulted in reduced constriction. Constriction to hypocapnia was also reduced following isocapnic alkaline solution. Since we previously demonstrated that constrictions to hypocapnia and isocapnic alkaline solution were endothelin-1 dependent, we tested whether the inhibition of hypocapnia- and isocapnic alkaline solution-induced constrictions following isocapnic alkaline solution was due to reduced endothelin-1 constriction. Endothelin-1 constriction was not reduced following isocapnic alkaline solution. Thus, constriction to isocapnic alkaline solution does not mimic constriction to hypocapnia. The results further suggest that the decreased constriction to isocapnic alkaline solution is due to blockade of endothelin-1 release, and that both hypocapnia and isocapnic alkaline solution share a common step in their endothelin-1 release pathways that can be inhibited by isocapnic alkaline solution.

Acute nitric oxide synthase inhibition and endothelin-1-dependent arterial pressure elevation.

Key evidence that endogenous nitric oxide (NO) inhibits the continuous, endothelin (ET)-1-mediated drive to elevate arterial pressure includes demonstrations that ET-1 mediates a significant component of the pressure elevated by acute exposure to NO synthase (NOS) inhibitors. This review examines the characteristics of this pressure elevation in order to elucidate potential mechanisms associated with the negative regulation of ET-1 by NO and, thereby, provide potential insight into the vascular pathophysiology underlying NO dysregulation. We surmise that the magnitude of the ET-1-dependent component of the NOS inhibitor-elevated pressure is (1) independent of underlying arterial pressure and other pressor pathways activated by the NOS inhibitors and (2) dependent on relatively higher NOS inhibitor dose, release of stored and de novo synthesized ET-1, and ETA receptor-mediated increased vascular resistance. Major implications of these conclusions include: (1) the marked variation of the ET-1-dependent component, i.e., from 0 to 100% of the pressure elevation, reflects the NO-ET-1 regulatory pathway. Thus, NOS inhibitor-mediated, ET-1-dependent pressure elevation in vascular pathophysiologies is an indicator of the level of compromised/enhanced function of this pathway; (2) NO is a more potent inhibitor of ET-1-mediated elevated arterial pressure than other pressor pathways, due in part to inhibition of intravascular pressure-independent release of ET-1. Thus, the ET-1-dependent component of pressure elevation in vascular pathophysiologies associated with NO dysregulation is of greater magnitude at higher levels of compromised NO.

Sensory nerves and transient receptor potential vanilloid 1 channels in CO(2) regulation of cerebrovascular tone.

This study investigated the involvement of sensory nerves and, in particular, neuronal transient receptor potential vanilloid (TRPV) 1 channels, in the CO(2)-mediated regulation of cerebrovascular tone. Basilar artery diameter and blood flow velocity in the ventral midbrain were determined in a rat cranial window preparation by digital imaging and laser-Doppler flowmetry, respectively. Superfusion of the basilar artery with capsaicin, a selective TRPV1 receptor agonist, caused a transient relaxation, consistent with acute desensitization of neuronal TRPV1 channels. Constriction to respiratory hypocapnia remained unaffected following capsaicin superfusion. Denervation of sensory nerves by repeated capsaicin injection of neonates also did not reduce the respiratory hypocapnia constriction of the basilar artery as well as the decreased flow velocity in the ventral midbrain in adults. These findings suggest that sensory nerves and, in particular, neuronal TRPV1 channels, do not play a role in respiratory hypocapnia constriction and decreased flow, at least in rat basilar artery and ventral midbrain.