George C. Wellman

Targeted Disruption of Kir2.1 and Kir2.2 Genes Reveals the Essential Role of the Inwardly Rectifying K + Current in K + -Mediated Vasodilation

The molecular bases of inwardly rectifying K(+) (Kir) currents and K(+)-induced dilations were examined in cerebral arteries of mice that lack the Kir2.1 and Kir2.2 genes. The complete absence of the open reading frame in animals homozygous for the targeted allele was confirmed. Kir2.1(-/-) animals die 8 to 12 hours after birth, apparently due to a complete cleft of the secondary palate. In contrast, Kir2.2(-/-) animals are viable and fertile. Kir currents were observed in cerebral artery myocytes isolated from control neonatal animals but were absent in myocytes from Kir2.1(-/-) animals. Voltage-dependent K(+) currents were similar in cells from neonatal control and Kir2.1(-/-) animals. An increase in the extracellular K(+) concentration from 6 to 15 mmol/L caused Ba(2+)-sensitive dilations in pressurized cerebral arteries from control and Kir2.2 mice. In contrast, arteries from Kir2.1(-/-) animals did not dilate when the extracellular K(+) concentration was increased to 15 mmol/L. In summary, Kir2.1 gene expression in arterial smooth muscle is required for Kir currents and K(+)-induced dilations in cerebral arteries.

Signaling between SR and plasmalemma in smooth muscle: sparks and the activation of Ca2+-sensitive ion channels.

Intracellular calcium ions are involved in the regulation of nearly every aspect of cell function. In smooth muscle, Ca2+ can be delivered to Ca2+-sensitive effector molecules either by influx through plasma membrane ion channels or by intracellular Ca2+ release events. Ca2+ sparks are transient local increases in intracellular Ca2+ that arise from the opening of ryanodine-sensitive Ca2+ release channels (ryanodine receptors) located in the sarcoplasmic reticulum. In arterial myocytes, Ca2+ sparks occur near the plasma membrane and act to deliver high (microM) local Ca2+ to plasmalemmal Ca2+-sensitive ion channels, without directly altering global cytosolic Ca2+ concentrations. The two major ion channel targets of Ca2+ sparks are Ca2+-activated chloride (Cl(Ca)) channels and large-conductance Ca2+-activated potassium (BK) channels. The activation of BK channels by Ca2+ sparks play an important role in the regulation of arterial diameter and appear to be involved in the action of a variety of vasodilators. The coupling of Ca2+ sparks to BK channels can be influenced by a number of factors including membrane potential and modulatory beta subunits of BK channels. Cl(Ca) channels, while not present in all smooth muscle, can also be activated by Ca2+ sparks in some types of smooth muscle. Ca2+ sparks can also influence the activity of Ca2+-dependent transcription factors and expression of immediate early response genes such as c-fos. In summary, Ca2+ sparks are local Ca2+ signaling events that in smooth muscle can act on plasma membrane ion channels to influence excitation-contraction coupling as well as gene expression.

Endothelium-Dependent Dilation of Feline Cerebral Arteries: Role of Membrane Potential and Cyclic Nucleotides

The objective of this study was to characterize the role of membrane potential and cyclic nucleotides in endothelium-dependent dilation of cerebral arteries. Middle cerebral arteries isolated from cats were depolarized and constricted in response to serotonin or when subjected to transmural pressures >50 mm Hg. Acetylcholine (ACh) and ADP caused vasodilation and a sustained, dose-dependent hyperpolarization of up to 20 mV in this artery. The membrane potential change preceded the vasodilation by ∼6 s. Hyperpolarizations and dilations to ACh and ADP did not occur in preparations without endothelium. The hyperpolarizations were abolished by ouabain (10−5 M), which also blocked the dilator response to ACh. However, dilations to ADP were unaffected by ouabain. Methylene blue (5 × 10−5 M), a guanylate cyclase inhibitor, had no effect on the responses to ACh or ADP in the presence or absence of ouabain. Cyclic guanosine monophosphate (cGMP) levels were not altered in cerebral arteries exposed to ACh or ADP. However, ADP did increase cyclic adenosine monophosphate levels in these blood vessels. We conclude that although membrane hyperpolarizations may be adequate to cause vasodilation, at least one other pathway of endothelium-dependent vasodilation also is present in feline cerebral arteries. Cyclic GMP does not appear to be involved in this alternate pathway of dilation.

Emergence of a r-type ca2+channel(cav2.3) contributes to cerebral artery constriction following subarachnoid hemorrhage

Cerebral aneurysm rupture and subarachnoid hemorrhage (SAH) inflict disability and death on thousands of individuals each year. In addition to vasospasm in large diameter arteries, enhanced constriction of resistance arteries within the cerebral vasculature may contribute to decreased cerebral blood flow and the development of delayed neurological deficits after SAH. In this study, we provide novel evidence that SAH leads to enhanced Ca2+ entry in myocytes of small diameter cerebral arteries through the emergence of R-type voltage-dependent Ca2+ channels (VDCCs) encoded by the gene CaV 2.3. Using in vitro diameter measurements and patch clamp electrophysiology, we have found that L-type VDCC antagonists abolish cerebral artery constriction and block VDCC currents in cerebral artery myocytes from healthy animals. However, 5 days after the intracisternal injection of blood into rabbits to mimic SAH, cerebral artery constriction and VDCC currents were enhanced and partially resistant to L-type VDCC blockers. Further, SNX-482, a blocker of R-type Ca2+ channels, reduced constriction and membrane currents in cerebral arteries from SAH animals, but was without effect on cerebral arteries of healthy animals. Consistent with our biophysical and functional data, cerebral arteries from healthy animals were found to express only L-type VDCCs (CaV 1.2), whereas after SAH, cerebral arteries were found to express both CaV 1.2 and CaV 2.3. We propose that R-type VDCCs may contribute to enhanced cerebral artery constriction after SAH and may represent a novel therapeutic target in the treatment of neurological deficits after SAH.

Inversion of neurovascular coupling by subarachnoid blood depends on large-conductance Ca2+-activated K+ (BK) channels

The cellular events that cause ischemic neurological damage following aneurysmal subarachnoid hemorrhage (SAH) have remained elusive. We report that subarachnoid blood profoundly impacts communication within the neurovascular unit—neurons, astrocytes, and arterioles—causing inversion of neurovascular coupling. Elevation of astrocytic endfoot Ca2+ to ∼400 nM by neuronal stimulation or to ∼300 nM by Ca2+ uncaging dilated parenchymal arterioles in control brain slices but caused vasoconstriction in post-SAH brain slices. Inhibition of K+ efflux via astrocytic endfoot large-conductance Ca2+-activated K+ (BK) channels prevented both neurally evoked vasodilation (control) and vasoconstriction (SAH). Consistent with the dual vasodilator/vasoconstrictor action of extracellular K+ ([K+]o), [K+]o <10 mM dilated and [K+]o >20 mM constricted isolated brain cortex parenchymal arterioles with or without SAH. Notably, elevation of external K+ to 10 mM caused vasodilation in brain slices from control animals but caused a modest constriction in brain slices from SAH model rats; this latter effect was reversed by BK channel inhibition, which restored K+-induced dilations. Importantly, the amplitude of spontaneous astrocytic Ca2+ oscillations was increased after SAH, with peak Ca2+ reaching ∼490 nM. Our data support a model in which SAH increases the amplitude of spontaneous astrocytic Ca2+ oscillations sufficiently to activate endfoot BK channels and elevate [K+]o in the restricted perivascular space. Abnormally elevated basal [K+]o combined with further K+ efflux stimulated by neuronal activity elevates [K+]o above the dilation/constriction threshold, switching the polarity of arteriolar responses to vasoconstriction. Inversion of neurovascular coupling may contribute to the decreased cerebral blood flow and development of neurological deficits that commonly follow SAH.

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization

Intracerebral (parenchymal) arterioles are morphologically and physiologically unique compared with pial arteries and arterioles. The ability of subarachnoid hemorrhage (SAH) to induce vasospasm in large-diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is controversial. Currently, little is known regarding the impact of SAH on parenchymal arterioles, which are critical for regulation of local and global cerebral blood flow. Here diameter, smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)), and membrane potential measurements were used to assess the function of intact brain parenchymal arterioles isolated from unoperated (control), sham-operated, and SAH model rats. At low intravascular pressure (5 mmHg), membrane potential and [Ca(2+)](i) were not different in arterioles from control, sham-operated, and SAH animals. However, raising intravascular pressure caused significantly greater membrane potential depolarization, elevation in [Ca(2+)](i), and constriction in SAH arterioles. This SAH-induced increase in [Ca(2+)](i) and tone occurred in the absence of the vascular endothelium and was abolished by the L-type voltage-dependent calcium channel (VDCC) inhibitor nimodipine. Arteriolar [Ca(2+)](i) and tone were not different between groups when smooth muscle membrane potential was adjusted to the same value. Protein and mRNA levels of the L-type VDCC Ca(V)1.2 were similar in parenchymal arterioles isolated from control and SAH animals, suggesting that SAH did not cause VDCC upregulation. We conclude that enhanced parenchymal arteriolar tone after SAH is driven by smooth muscle membrane potential depolarization, leading to increased L-type VDCC-mediated Ca(2+) influx.

Store-Operated Ca2+ Entry Activates the CREB Transcription Factor in Vascular Smooth Muscle

Ca2+-regulated gene transcription is a critical component of arterial responses to injury, hypertension, and tumor-stimulated angiogenesis. The Ca2+/cAMP response element binding protein (CREB), a transcription factor that regulates expression of many genes, is activated by Ca2+-induced phosphorylation. Multiple Ca2+ entry pathways may contribute to CREB activation in vascular smooth muscle including voltage-dependent Ca2+ channels and store-operated Ca2+ entry (SOCE). To investigate a role for SOCE in CREB activation, we measured CREB phosphorylation using immunofluorescence, intracellular Ca2+ levels using a fluorescence resonance energy transfer (FRET)–based Cameleon indicator, and c-fos transcription using RT-PCR. In this study, we report that SOCE activates CREB in both cultured smooth muscle cells and intact arteries. Depletion of intracellular Ca2+ stores with thapsigargin increased nuclear phospho-CREB levels, intracellular Ca2+ concentration, and transcription of c-fos. These effects were abolished by inhibiting SOCE through lowering extracellular Ca2+ concentration or by application of 2-aminoethoxydiphenylborate and Ni2+. Inhibition of Ca2+ influx through voltage-dependent Ca2+ channels using nimodipine partially blocked intact artery responses, but was without effect in cultured smooth muscle cells. Our findings indicate that Ca2+ entry through store-operated Ca2+ channels leads to CREB activation, suggesting that SOCE contributes to the regulation of gene expression in vascular smooth muscle.

Flow-dependent dilation in a resistance artery still occurs after endothelium removal.

Infusion of physiological saline solution into the lumen of a tonically contracted resistance artery in vitro caused active relaxation. After endothelium removal by rubbing, confirmed by scanning electron microscopy and loss of the relaxation response to acetylcholine (1 microM), flow relaxation was reduced from a mean of 70% to 37%. The latter change was significant (p less than 0.01). It is concluded that flow-relaxation in the resistance artery of the rabbit originates from both the tunica intima and the media.

Oxyhemoglobin-Induced Suppression of Voltage-Dependent K+ Channels in Cerebral Arteries by Enhanced Tyrosine Kinase Activity

Cerebral vasospasm following aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences. Oxyhemoglobin (oxyhb) has been implicated in SAH-induced cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca2+-selective and K+-selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here we provide novel evidence that oxyhb selectively decreases 4-aminopyridine sensitive, voltage-dependent K+ channel (Kv) currents by ≈30% in myocytes isolated from rabbit cerebral arteries but did not directly alter the activity of voltage-dependent Ca2+ channels or large conductance Ca2+-activated (BK) channels. A combination of tyrosine kinase inhibitors (tyrphostin AG1478, tyrphostin A23, tyrphostin A25, genistein) abolished both oxyhb-induced suppression of Kv channel currents and oxyhb-induced constriction of isolated cerebral arteries. The Kv channel blocker 4-aminopyridine also inhibited oxyhb-induced cerebral artery constriction. The observed oxyhb-induced decrease in Kv channel activity could represent either channel block, or a decrease in Kv channel density on the plasma membrane. To explore whether oxyhb altered trafficking of Kv channels to the plasma membrane, we used an antibody generated against an extracellular epitope of Kv1.5 channels. In the presence of oxyhb, staining of Kv1.5 on the plasma membrane surface was markedly reduced. Furthermore, oxyhb caused a loss of spatial distinction between staining with Kv1.5 and the general anti-phosphotyrosine antibody PY-102. We propose that oxyhb-induced suppression of Kv currents occurs via a mechanism involving enhanced tyrosine kinase activity and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH.

Aminergic histofluorescence and contractile responses to transmural electrical field stimulation and norepinephrine of human middle cerebral arteries obtained promptly after death.

The responses of cerebral arteries to catecholamines and sympathetic nerve stimulation show wide variation between animal species. We examined the catecholaminergic histofluorescence and the contractile responses elicited by transmural electrical field stimulation and norepinephrine (NE) in proximal segments of human middle cerebral artery (MCA) obtained during autopsy. Twenty-four percent of the specimens were obtained within 2 hours and 76% within 4 hours of death. A moderately dense catecholaminergic histofluorescence was seen in all segments of human MCA using the glyoxylic acid technique, counterstained with pontamine sky blue. However, only seven of 35 (20%) MCA segments tested showed tetrodotoxin-blocked transmural electrical field stimulation contractions, and all of these were harvested within 4 hours of death. The responses were mostly seen in the most proximal MCA segments and, at 32 Hz, only achieved 6 +/- 1% of the maximal tissue contraction. NE caused two distinct responses in human MCA segments. At low concentrations, it acts via an alpha-like adrenoreceptor to cause contractions 20 +/- 3% of the maximal tissue response. The NE ED50s for the three successive segments were not different from each other; the value for the most-proximal segment was 7.9 +/- 0.2 x 10(-7) M. At concentrations above 10(-5) M, this catecholamine acts on low-affinity sites resistant to alpha-adrenergic antagonists causing contractions that at 10(-3) M reach 52 +/- 5% of the maximal tissue response.(ABSTRACT TRUNCATED AT 250 WORDS)