Jane A. Madden

Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation

SUMMARY How vertebrate blood vessels sense acute hypoxia and respond either by constricting (hypoxic vasoconstriction) or dilating (hypoxic vasodilation) has not been resolved. In the present study we compared the mechanical and electrical responses of select blood vessels to hypoxia and H2S, measured vascular H2S production, and evaluated the effects of inhibitors of H2S synthesis and addition of the H2S precursor, cysteine, on hypoxic vasoconstriction and hypoxic vasodilation. We found that: (1) in all vertebrate vessels examined to date, hypoxia and H2S produce temporally and quantitatively identical responses even though the responses vary from constriction (lamprey dorsal aorta; lDA), to dilation (rat aorta; rA), to multi-phasic (rat and bovine pulmonary arteries; rPA and bPA, respectively). (2) The responses of lDA, rA and bPA to hypoxia and H2S appear competitive; in the presence of one stimulus, the response to the other stimulus is substantially or completely eliminated. (3) Hypoxia and H2S produce the same degree of cell depolarization in bPA. (4) H2S is constitutively synthesized by lDA and bPA vascular smooth muscle. (5) Inhibition of H2S synthesis inhibits the hypoxic response of lDA, rA, rPA and bPA. (6) Addition of the H2S precursor, cysteine, doubles hypoxic contraction in lDA, prolongs contraction in bPA and alters the re-oxygenation response of rA. These studies suggest that H2S may serve as an O2 sensor/transducer in the vascular responses to hypoxia. In this model, the concentration of vasoactive H2S in the vessel is governed by the balance between endogenous H2S production and its oxidation by available O2.

High Blood Pressure Upregulates Arterial L-Type Ca2+ Channels Is Membrane Depolarization the Signal?

Long-lasting Ca2+ (CaL) channels of the Cav1.2 gene family contribute to the pathogenesis of abnormal arterial tone in hypertension. The physiological stimulus that enhances CaL channel current in the vascular smooth muscle cells (VSMCs) remains unknown. The present study investigated if high blood pressure triggers an upregulation of vascular CaL channel protein. Rat aortae were banded between the origins of the left renal (LR) and right renal (RR) arteries to selectively elevate blood pressure in the proximal RR arteries. After 2 days, the immunoreactivity on Western blots corresponding to the pore-forming &agr;1C subunit of the CaL channel was increased 3.25-fold in RR compared with LR arteries. This finding persisted at 28 days and was associated with abnormal Ca2+-dependent tone and higher CaL currents in the VSMCs exposed to high pressure. Based on microelectrode studies indicating that RR arteries were depolarized compared with LR arteries, further studies examined if membrane depolarization, an inherent response of VSMCs to high blood pressure, increased &agr;1C expression. Isolated rat renal arteries were cultured for 2 days in low K+ (4 mmol/L) or depolarizing high K+ (30 mmol/L) media. Arteries preconditioned in high K+ showed a 5.47-fold increase in &agr;1C expression, enhanced CaL channel current, and elevated Ca2+-dependent tone. These findings provide the first direct evidence that high blood pressure upregulates the CaL channel &agr;1C subunit in VSMCs in vivo and suggest that membrane depolarization is a potential signal involved in this interaction that may contribute to the development of abnormal vascular tone.

Voltage‐gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels

Voltage‐gated potassium (KV) channels represent an important dilator influence in the cerebral circulation, but the composition of these tetrameric ion channels remains unclear. The goals of the present study were to evaluate the contribution of KV1 family channels to the resting membrane potential and diameter of small rat cerebral arteries, and to identify the α‐subunit composition of these channels using patch‐clamp, molecular and immunological techniques. Initial studies indicated that 1 μmol l−1 correolide (COR), a specific antagonist of KV1 channels, depolarized vascular smooth muscle cells (VSMCs) in pressurized (60 mmHg) cerebral arteries from ‐55 ± 1 mV to ‐34 ± 1 mV, and reduced the resting diameter from 152 ± 15 μm to 103 ± 20 μm. In patch clamped VSMCs from these arteries, COR‐sensitive KV1 current accounted for 65 % of total outward KV current and was observed at physiological membrane potentials. RT‐PCR identified mRNA encoding each of the six classical KV1 α‐subunits, KV1.1‐1.6, in rat cerebral arteries. However, only the KV1.2 and 1.5 proteins were detected by Western blot. The expression of these proteins in VSMCs was confirmed by immunocytochemistry and co‐immunoprecipitation of KV1.2 and 1.5 from VSMC membranes suggested KV1.2/1.5 channel assembly. Subsequently, the pharmacological and voltage‐sensitive properties of KV1 current in VSMCs were found to be consistent with a predominant expression of KV1.2/1.5 heterotetrameric channels. The findings of this study suggest that KV1.2/1.5 heterotetramers are preferentially expressed in rat cerebral VSMCs, and that these channels contribute to the resting membrane potential and diameter of rat small cerebral arteries.

Effect of cocaine and cocaine metabolites on cerebral arteries in vitro

Cocaine has pronounced peripheral vasoconstrictor effects. Despite the short half life of cocaine in the body these effects are relatively long-lived. The role of cocaine metabolites in vasoconstriction attributed to cocaine has not been reported. We evaluated the contractile ability of cocaine and its major metabolites in isolated cat cerebral arteries. The primary cocaine metabolite, benzoylecgonine was a potent contractile agent, causing a 50% decrease in cross sectional area at 10(-5) M. This was less than caused by serotonin, but greater than caused by norepinehrine. Ecgonine and cocaine were less active contractile agents than was benzoylecgonine, and ecgonine methyl ester was a mild relaxant.

Hypoxic pulmonary vasodilation: a paradigm shift with a hydrogen sulfide mechanism

Hypoxic pulmonary vasoconstriction (HVC), an intrinsic and assumed ubiquitous response of mammalian pulmonary blood vessels, matches regional ventilation to perfusion via an unknown O(2)-sensing mechanism. Global pulmonary hypoxia experienced by individuals suffering from chronic obstructive pulmonary disease or numerous hypoventilation syndromes, including sleep apnea, often produces maladaptive pulmonary hypertension, but pulmonary hypertension is not observed in diving mammals, where profound hypoxia is routine. Here we examined the response of cow and sea lion pulmonary arteries (PA) to hypoxia and observed the expected HVC in the former and a unique hypoxic vasodilation in resistance vessels in the latter. We then used this disparate response to examine the O(2)-sensing mechanism. In both animals, exogenous H(2)S mimicked the vasoactive effects of hypoxia in isolated PA. H(2)S-synthesizing enzymes, cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfur transferase, were identified in lung tissue from both animals by one-dimensional Western blot analysis and immunohistochemistry. The relationship between H(2)S production/consumption and O(2) was examined in real time by use of amperometric H(2)S and O(2) sensors. H(2)S was produced by sea lion and cow lung homogenate in the absence of O(2), but it was rapidly consumed when O(2) was present. Furthermore, consumption of exogenous H(2)S by cow lung homogenate, PA smooth muscle cells, and heart mitochondria was O(2) dependent and exhibited maximal sensitivity at physiologically relevant Po(2) levels. These studies show that HVC is not an intrinsic property of PA and provide further evidence for O(2)-dependent H(2)S metabolism in O(2) sensing.

Cocaine and benzoylecgonine constrict cerebral arteries by different mechanisms.

This study was designed to determine possible mechanisms underlying the vasoconstrictor activity of cocaine and its principal metabolite, benzoylecgonine (BE) in cat isolated cerebral arteries. The arteries constricted significantly in response to single doses of cocaine, BE and norepinephrine (NE; (P < 0.05). After 6-OHDA treatment to remove adrenergic nerve endings, NE-induced constrictions were essentially unchanged from those before treatment. Denervated arteries exposed to cocaine dilated significantly (P < 0.05) but those exposed to BE constricted as much as before denervation. Following exposure to prazosin and yohimbine, arterial constrictions to NE and cocaine were significantly reduced from control (P < 0.05) but the BE-induced constriction was unchanged. Ryanodine eliminated the cocaine-induced contraction (P < 0.05) whereas verapamil eliminated the BE response (P < 0.05). These data suggest that while cocaines vasoconstrictor action may be significantly mediated through adrenergic transmission, BE may act through a mechanism involving calcium (Ca2+) channels. Cocaine levels peak and decline in the body more rapidly than BE levels which can remain detectable for days. This study suggests there may also be different pharmacological mechanisms as well as temporal differences underlying the vasoreactivity of these two substances. Our findings may have implications for pharmacological management of cocaine-induced toxic vascular events.

Cellular mechanism of force development in cat middle cerebral artery by reducedPCO2

These studies were undertaken to determine the effect of reducing aPCO2 below physiological levels on cat middle cerebral artery. Upon reduction ofPCO2 from 37 to 14 torr (pH 7.4) we observed membrane depolarization and force development. ReducingPCO2 decreased the slope of theEm vs. log [K]o curve and increased the slope of the steady-state I/V relationship suggesting that the change inEm was due to reduction of outward K+ conductance (gk). Elevation of pH from 7.37 to 7.6 had a very similar effect on these cerebral arterial muscle cells, depolarizing the muscle membrane (reducing theEm vs. log [K]o curve) and increasing the slope of the I/V relationship to statistically equivalent values as reduction ofPCO2. ReturningPCO2 from 14 to 37 torr rapidly relaxed these preparations, but only transiently. This relaxation was followed by a rebound contraction within 3 min, demonstrating a transient nature for the action of elevatingPCO2 in cerebral arteries. The response to changing pHo followed a slower time course but did not change with time. These studies demonstrate that both elevated pHo and reducedPCO2 activate cerebral arterial muscle by a mechanism which includes reduction ingk. However, it can not be determined if these similar responses and reduction, ofgk are mediated by changing pHi or mediated through different mechanisms. It is possible that pHo andPCO2 can modify cerebral arterial tone by direct mechanisms and not necesarily by their effect on pHi. It is clear, however, that reduction ofPCO2 and elevation of pHo both activate cerebral arterial muscle by a mechanism which includes reduction ofgk.

Integrin signaling, free radicals, and tyrosine kinase mediate flow constriction in isolated cerebral arteries.

Isolated, cannulated, and pressurized (100 mmHg) middle cerebral arteries from adult cats were perfused intraluminally at rates from 0 to 4 ml/min with heated and gassed physiological saline solution. An electronic system held pressure constant by changing outflow resistance. The arteries constricted 18.1 ± 0.95% in response to flow and depolarized from -54 ± 0.51 to -40 ± 1.26 mV ( P < 0.05). Constriction was independent of a functional endothelium but was eliminated by superoxide dismutase or tyrosine kinase inhibitors. Luminal perfusion with a synthetic extracellular matrix Arg-Gly-ASP (RGD) peptide that binds with integrin significantly reduced constriction to flow. Neither reducing intraluminal pressure nor increasing tone or shear stresses altered constriction to flow. Flow-induced constriction did not impede the ability of the arteries to dilate to hypercapnia, and inhibiting flow-induced constriction did not alter contractile responses to other agonists. These data suggest that, in vitro, middle cerebral arteries constrict to flow through a mechanism involving free radicals and tyrosine kinase and that flow shear stresses resulting in constriction are transduced by integrin signaling.

Seizures Induced by the Cocaine Metabolite Benzoylecgonine in Rats

Summary: The half‐life (t1/2) of cocaine is relatively short, but some of the consequences of its use, such as seizures and strokes, can occur hours after exposure. This led us to hypothesize that a metabolite of cocaine may be responsible for some of those delayed sequelae. We evaluated the potential of the major metabolite of cocaine, benzoylecgonine (BE), to cause seizures. Two separate equimolar doses (0.2 and 0.4 μmol) of either cocaine or BE were injected ventricularly in unanesthetized juvenile rats. Treated rats were then evaluated for incidence, latency, and seizure pattern or for locomotor activity in animals without seizures. BE‐Induced seizures occurred more frequently and had significantly longer latencies than those induced by equimolar amounts of cocaine. Whereas cocaine‐induced seizures were best characterized as brief, generalized, and tonic and resulted in death, those induced by BE were prolonged, often multiple and mixed in type, and rarely resulted in death. Electrical recordings from the hippocampus showed a rhythmic progression in EEG frequency and voltage with clinical seizure expression. BE‐Injected rats that did not have seizures had significantly more locomotor activity than cocaine‐injected animals without seizures. The finding that cocaineand BE‐induced seizures differ in several respects suggests more than one mechanism for cocaine‐induced seizures and emphasizes the importance of a cocaine metabolite, BE.

Precursors and inhibitors of hydrogen sulfide synthesis affect acute hypoxic pulmonary vasoconstriction in the intact lung

The effects of hydrogen sulfide (H(2)S) and acute hypoxia are similar in isolated pulmonary arteries from various species. However, the involvement of H(2)S in hypoxic pulmonary vasoconstriction (HPV) has not been studied in the intact lung. The present study used an intact, isolated, perfused rat lung preparation to examine whether adding compounds essential to H(2)S synthesis or to its inhibition would result in a corresponding increase or decrease in the magnitude of HPV. Western blots performed in lung tissue identified the presence of the H(2)S-synthesizing enzymes, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), but not cystathionine β-synthase (CBS). Adding three H(2)S synthesis precursors, cysteine and oxidized or reduced glutathione, to the perfusate significantly increased peak arterial pressure during hypoxia compared with control (P < 0.05). Adding α-ketoglutarate to enhance the 3-MST enzyme pathway also resulted in an increase (P < 0.05). Both aspartate, which inhibits the 3-MST synthesis pathway, and propargylglycine (PPG), which inhibits the CSE pathway, significantly reduced the increases in arterial pressure during hypoxia. Diethylmaleate (DEM), which conjugates sulfhydryls, also reduced the peak hypoxic arterial pressure at concentrations >2 mM. Finally, H(2)S concentrations as measured with a specially designed polarographic electrode decreased markedly in lung tissue homogenate and in small pulmonary arteries when air was added to the hypoxic environment of the measurement chamber. The results of this study provide evidence that the rate of H(2)S synthesis plays a role in the magnitude of acute HPV in the isolated perfused rat lung.