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(Circulation Research. 2001;88:359.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Chronic Nicotine Alters NO Signaling of Ca²⁺ Channels in Cerebral Arterioles

Volodymyr Gerzanich, Fangyi Zhang, G. Alexander West, J. Marc Simard

From the Departments of Neurosurgery (V.G., J.M.S.) and Physiology (J.M.S.), University of Maryland School of Medicine, Baltimore, Md; Division of Neurosurgery (F.Z.), University of Texas at San Antonio Health Science Center, San Antonio, Tex; and Department of Neurological Surgery (G.A.W.), University of Washington School of Medicine, Seattle, Wash.

Correspondence to Dr J. Marc Simard, Department of Neurosurgery, University of Maryland School of Medicine, 22 S Greene St, Baltimore, MD 21201-1595. E-mail msimard{at}surgery1.umaryland.edu

	Abstract

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Abstract—Smoking is a major health hazard with proven deleterious effects on the cerebral circulation, including a decrease in cerebral blood flow and a high risk for stroke. To elucidate cellular mechanisms for the vasoconstrictive and pathological effects of nicotine, we used a nystatin-perforated patch-clamp technique to study Ca²⁺ channels and Ca²⁺-activated K⁺ (BK) channels in smooth muscle cells isolated from cerebral lenticulostriate arterioles of rats chronically exposed to nicotine (4.5 mg/kg per day of nicotine free base, 15 to 22 days via osmotic minipump). Two major effects were observed in cells from nicotine-treated animals compared with controls. First, Ca²⁺ channels were upregulated (0.48±0.03 pS/pF [20 cells] versus 0.35±0.01 pS/pF [31 cells], P<0.005) and BK channels were downregulated (12±3 pA/pF [14 cells] versus 34±7 pA/pF [14 cells], P<0.05), mimicking the effect of an apparent decrease in bioavailability of endogenous NO. Second, normal downregulation of Ca²⁺ channels by exogenous NO (sodium nitroprusside [SNP], 100 nmol/L) and cGMP (8-bromo-cGMP, 0.1 mmol/L) was absent, whereas normal upregulation of BK channels by these agents was preserved, suggesting block of NO signaling downstream of cGMP-dependent protein kinase. In pial window preparations, chronic nicotine blunted NO-induced vasodilation of pial vessels and the increase in cortical blood flow measured by laser-Doppler flowmetry, demonstrating the importance of Ca²⁺ channel downregulation in NO-induced vasorelaxation. These findings elucidate a new pathophysiological mechanism involving altered Ca²⁺ homeostasis in cerebral arterioles that may predispose to stroke.

Key Words: Ca²⁺ channel • vascular smooth muscle • nicotine • nitric oxide • cerebral arteriole

	Introduction

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Tobacco smoke, a health hazard most widely associated with cancer, is also a significant risk factor for stroke.¹ Chronic exposure to tobacco smoke or to nicotine, a major vasoactive substance in tobacco, causes cerebral vasoconstriction and decreases cortical blood flow (CBF).^{2 3} Outside of the CNS, nicotine is well known to enhance vasoconstriction by impairing endothelium-dependent and endothelium-independent vasodilation.^{4 5} Vasoconstrictive effects of tobacco smoke and nicotine have been attributed to inhibition of endothelial NO synthase (NOS)⁶ or impaired NO signaling,^{4 7} which is critical for regulating cerebrovascular tone.⁸

NO induces vasorelaxation in part by decreasing Ca²⁺ influx through smooth muscle L-type Ca²⁺ channels, via both voltage-dependent and voltage-independent mechanisms. Voltage-dependent inhibition arises from cGMP-dependent protein kinase (PKG) causing an increase in open probability of Ca²⁺-activated K⁺ (BK) channels,^{9 10} which polarize the cell and deactivate Ca²⁺ channels. Voltage-independent inhibition arises from PKG phosphorylating the Ca²⁺ channel itself or, more likely, a closely related regulatory phosphoprotein, thereby decreasing its open probability independently of voltage.^{11 12}

Given the important regulatory role of NO on ion channels, we postulated that impaired NO signaling expected with nicotine would increase availability of Ca²⁺ channels and decrease availability of BK channels in smooth muscle cells. To test this hypothesis, we examined effects of chronic nicotine on availability of functional Ca²⁺ channels and K⁺ channels in lenticulostriate arteriolar smooth muscle cells (LSA-SMCs) of rats, vessels that in humans are preferentially involved in stroke.^{13 14} Also, to validate findings made on isolated cells, we examined the effects of chronic nicotine on vasomotor tone and on responses to NO in pial vessels in intact animals. Here we report that chronic exposure to nicotine, at concentrations comparable with those in humans who actively smoke cigarettes,¹⁵ caused 2 distinct effects related to smooth muscle ion channel regulation. First, nicotine increased availability of Ca²⁺ channels and decreased availability of Ca²⁺-activated K⁺ channels in cerebral arterioles. Secondly, it altered NO signaling of L-type Ca²⁺ channels by a mechanism heretofore undescribed, causing a block of normal downregulation of Ca²⁺ channels by NO and cGMP without altering normal upregulation of Ca²⁺-activated K⁺ channels by NO and cGMP. Moreover, the significance of these ion channel effects was corroborated by showing reduced pial vasorelaxation in response to NO in animals chronically exposed to nicotine. These novel effects of nicotine, not previously reported for any chemical agent, provide new insight into the signaling mechanism used by NO to achieve vasorelaxation and provide a mechanism for altered Ca²⁺ homeostasis leading to pathological effects that may predispose to stroke.

	Materials and Methods

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Animal Model
Female Wistar-Kyoto rats, 220 to 300 g, were implanted with osmotic minipumps (Alzet 2002, Alza Corp) that delivered 0.9% NaCl without or with nicotine tartrate (Sigma; 4.5 mg/kg per day of nicotine free base) for 15 to 22 days, yielding plasma levels of nicotine (500 nmol/L)^{16 17} comparable with those in smokers.¹⁵ Animals exposed to nicotine showed normal growth, weight, and blood pressure.

Laser Doppler Flowmetry (LDF) and Pial Vessel Diameter
Experiments with either saline pump– or nicotine pump–implanted rats were blinded to the experimenters. An animal was anesthetized with 1% halothane. The right femoral artery and vein were cannulated for monitoring blood pressure, blood gas sampling, and intravenous drug administration. The animal was tracheostomized, immobilized with tubocuraine chloride (1 mg/kg IV), and mechanically ventilated to maintain physiological blood gas tensions. Rectal temperature was maintained at 37°C. The animal was secured in a stereotactic frame, and the skull was exposed through a longitudinal incision for preparation of a closed cranial window.¹⁸ The cranial window was continuously superfused at 0.5 mL/min with artificial cerebrospinal fluid equilibrated to maintain normal cerebrospinal fluid pH and gas tension as well as intracranial pressure. Pial vessel diameter was continuously monitored using a videomicroscopy system (Microcirculation Research Institute, Texas A&M University Health Science Center). CBF was measured simultaneously in the contralateral hemisphere using LDF (TSI Inc) via an open cranial window.¹⁹ The laser-Doppler probe (0.8 mm) was placed 0.5 mm above the pial surface, directed away from large pial vessels. Probe position and reactivity of the preparation were tested by assessing responses to hypercapnia. Measurements of vessel diameter and LDF (arbitrary units) are given as mean±SE. Statistical significance was assessed using a paired Student t test.

Single-Cell Preparation
An animal was killed by intraperitoneal injection of an overdose of sodium pentobarbital (100 mg/kg), was exsanguinated, and was subjected postmortem to transcardiac perfusion at constant hydrostatic pressure of 100 cm H₂O for 15 minutes. This perfusion pressure was sufficient for reliable perfusion of small cerebral arterioles and has been shown not to impair vascular endothelial or smooth muscle cell function.²⁰ After harvesting the brain, lenticulostriate arterioles were dissected from the internal carotid and middle cerebral arteries under magnification at room temperature. A yield of 15 to 25 microvessels could usually be harvested during 1 to 1.5 hours. Arterioles were subsequently processed to isolate single cells using the enzymatic digestion procedure previously used in this laboratory,²¹ except that the second enzyme step was omitted. Cells were stored at 4°C in modified KB solution and were studied within 10 hours of harvest. Only cells having a distinctive ring-shaped appearance were found to give consistent, high-level Ca²⁺ channel activity, and thus only such cells were used for this study. For cell isolation from nicotine-exposed animals, nicotine (0.5 µmol/L) was included in all solutions used for perfusion, dissection of vessels, cell isolation, storage, and patch-clamp experiments.

Patch Clamp
The method used for perforated patch recording in this laboratory has been described.²¹ Experiments were carried out at room temperature. Membrane currents were measured during step (200-ms) or ramp (-60 to +50 mV, 0.45 mV/ms) pulses from a holding potential of -60 mV. For Ca²⁺ channel recordings, the bath solution contained (in mmol/L) TEA·Cl 130, MgCl₂ 1, BaCl₂ 10, HEPES 10, glucose 12.5, and 4-aminopyridine 2 (pH 7.2 with TEA·OH), and the pipette solution contained CsCl 130, MgCl₂ 8, and HEPES 10 (pH 7.35 with CsOH plus nystatin). For recording Ca²⁺-activated K⁺ channels, the bath solution contained (in mmol/L) NaCl 140, KCl 5, MgCl₂ 2, HEPES 10, and glucose 12.5 (pH 7.4 with NaOH), and the pipette solution contained KCl 145, MgCl₂ 2, CaCl₂ 3.66, EGTA 5, HEPES 10, and glucose 10 (pH 7.2 with KOH).

Data Analysis
To quantify Ca²⁺ channel availability, current-voltage data from individual cells between -40 V and +40 mV were fit to the Boltzmann function:

where I is current; E is membrane potential; g_max is the maximum conductance at positive potentials; k and E_1/2 are the steepness and midpoint potential of activation, respectively; and E_rev is the extrapolated reversal potential for the chord conductance. Ca²⁺ channel availability was then calculated as normalized maximum conductance, ng_max, obtained by dividing g_max by cell capacitance.

Data were fit to the equation using the iterative nonlinear, least-squares method of Marquardt-Levenberg (Origin 6.0, Microcal). Statistical comparisons were evaluated using Student t test. All data are given as mean±SE.

	Results

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We first characterized the Ca²⁺ channels in LSA-SMCs because they had not been previously described. When recorded with 10 mmol/L Ba²⁺ as the charge carrier, the macroscopic inward currents showed kinetic and voltage-dependent properties that are typical for L-type Ca²⁺ channels (Figures 1A and 1Ca). The activating dihydropyridine, Bay k8644, doubled the current (Figure 1Cb), and the blocking dihydropyridine, nifedipine, blocked most of the current (Figure 1Cc). Recordings of cell-attached patches studied with 40 mmol/L Ba²⁺ plus Bay k8644 in the pipette solution revealed single-channel inward currents (Figure 1E, a through c) with a slope conductance of 22 pS (Figure 1F, closed circles). Together, these findings are consistent with previous reports on Ca²⁺ channels in other vascular smooth muscle cells^{22 23} and confirm that LSA-SMCs express predominantly a single class of L-type, dihydropyridine-sensitive Ca²⁺ channels. As previously observed in cerebral smooth muscle, no evidence was found for T-type Ca²⁺ channels in these cells.²⁴

View larger version (35K): [in this window] [in a new window]   Figure 1. Chronic nicotine upregulates Ca2+ channels in LSA-SMCs. A and B, Ca2+ channel currents during step pulses from -40 to +40 mV in LSA-SMCs from a control animal (A, 12 pF) and from a chronic nicotine animal (B, 13 pF). C and D, Current-voltage curves recorded during ramp pulses in LSA-SMCs from a control animal (C, 9 pF) and from a chronic nicotine animal (D, 14 pF) before (a) and after 1 µmol/L Bay k8644 (b) and 1 µmol/L nifedipine (c). E, Single-channel currents, with inward cationic currents plotted downward, in cell-attached patches from a control animal (a through c) and from a chronic nicotine animal (d through f), recorded at E=-30 mV (a and d), -40 mV (b and e), and -50 mV (c and f), with Bay k8644 and 40 mmol/L Ba2+ in the pipette solution. F, Open-channel current vs membrane potential showing 22-pS conductance in 5 membrane patches from control (•) and 5 membrane patches from chronic nicotine () animals. G, Average (±SE) current-voltage curves recorded during ramp pulses in LSA-SMCs from control (a, 25 cells; capacitance, 11.8±0.6 pF) and from chronic nicotine (b, 14 cells; capacitance, 11.2±0.8 pF) animals. H, Normalized values for Ca2+ channel availability in LSA-SMCs were obtained by fitting to a Boltzmann equation to obtain gmax and dividing by cell capacitance. Values from control animals (CONT) and chronic nicotine (NIC) animals, 0.35±0.01 pS/pF (31 cells) and 0.48±0.03 pS/pF (20 cells) respectively, were significantly different by t test (P<0.005). Fit to Boltzmann equation gave voltage-dependent parameters of E1/2=-3.6±0.8 and -2.4±1.1 mV and k=5.6±0.2 and 5.1±0.2 mV for the control and nicotine groups, respectively, which were not significantly different by t test (P>0.05).

In cells from rats chronically exposed to nicotine, the biophysical and pharmacological properties of the Ca²⁺ channel currents were indistinguishable from those of controls. Macroscopic inward currents showed kinetic and voltage-dependent properties typical for L-type Ca²⁺ channels (Figures 1B and 1Da). The activating dihydropyridine, Bay k8644, increased the current (Figure 1Db), and the blocking dihydropyridine, nifedipine, blocked it (Figure 1Dc). Recordings of cell-attached patches revealed single-channel inward currents (Figure 1E, d–f) with a slope conductance of 22 pS (Figure 1F, open circles).

However, in cells from rats chronically exposed to nicotine, the magnitude of the Ca²⁺ channel current was appreciably larger than in cells from control animals (Figure 1G). Current-voltage curves for individual cells were fit to a Boltzmann function (Equation) to quantify channel availability, revealing that normalized values in cells from nicotine-treated rats were significantly elevated (Figure 1H), although they showed no change in voltage dependence (see legend, Figure 1). Thus, chronic exposure to nicotine significantly augmented availability of Ca²⁺ channels without causing any change in kinetic or voltage-dependent properties, single-channel conductance, or pharmacological response to dihydropyridines.

In smooth muscle, outward K⁺ currents regulate Ca²⁺ channels by polarizing the cell and deactivating Ca²⁺ channels. We recorded outward currents in LSA-SMCs at different voltages (Figure 2A). The outward current was dominated by BK channels, as indicated by its insensitivity to glibenclamide, minimal block by 4-aminopyridine, and high sensitivity to iberiotoxin and charybdotoxin (Figure 2C).²⁵ In cells from chronic nicotine rats, electrophysiological and pharmacological properties, including kinetics, voltage dependence (Figure 2B), and sensitivity to iberiotoxin and charybdotoxin (Figure 2D), were similar to those of controls, which suggests no change in types of channels expressed. However, the magnitude of the current was significantly smaller in cells from chronic nicotine animals (Figure 2E). Thus, chronic exposure to nicotine resulted not only in upregulation of Ca²⁺ channels but also in downregulation of BK channels.

View larger version (29K): [in this window] [in a new window]   Figure 2. Chronic nicotine downregulates Ca2+-activated K+ (BK) channels in LSA-SMCs. A and B, Outward K+ channel currents during 200-ms (A) or 500-ms (B) step pulses from 0 to +100 mV in 10-mV steps (holding potential, -60 mV), in LSA-SMCs from a control animal (A, 12 pF) and from a chronic nicotine animal (B, 10 pF). C, Outward K+ channel currents during 200-ms step pulses to +80 mV (holding potential, -60 mV) in LSA-SMCs from a control animal in control conditions (a) and in the presence of 10 µmol/L glibenclamide (b), 5 mmol/L 4-aminopyridine (c), and 100 nmol/L iberiotoxin (d). D, Outward K+ channel currents during 200-ms step pulses to +80 mV (holding potential, 0 mV) in LSA-SMCs from a chronic nicotine animal in control conditions (a) and in the presence of 100 nmol/L iberiotoxin (b). E, Normalized values (end of 200-ms pulses to +80 mV, holding potential of 0 mV) for availability of BK channels in LSA-SMCs from control (CONT) and chronic nicotine (NIC) animals, 34±7 pA/pF (14 cells) and 12±3 pA/pF (14 cells), respectively, were significantly different by t test (P<0.01).

NO is known to downregulate Ca²⁺ channels and upregulate BK channels.^{9 10 11 12} We thus interpreted our findings of augmented Ca²⁺ channel availability and decreased BK channel availability with nicotine as being consistent with an apparent decrease in bioavailability of endogenous NO. This hypothesis accorded with our recent finding that block of endogenous NOS activity, as well as acute endothelial injury, results in upregulation of Ca²⁺ channels in smooth muscle cells.²¹ Because we also found in the same report that block of endogenous NO augments the apparent efficacy of exogenous NO in downregulating Ca²⁺ channels, we sought here to determine whether chronic nicotine also would cause an increase in apparent efficacy of NO.

Cells were studied using 100 nmol/L of the NO donor sodium nitroprusside (SNP), a concentration that causes maximum downregulation of Ca²⁺ channel currents.²¹ The effects of SNP in LSA-SMCs from control animals were comparable with previous observations in vascular smooth muscle, with a gradual decrease in whole-cell current (Figure 3C, closed circles) and no effect on voltage dependence (Figure 3A). The effect of NO donor on Ca²⁺ channel currents in cells from chronic nicotine rats was unexpected. In contrast to downregulation observed in cells from control animals (Figures 3A and 3C, closed circles), from other smooth muscle preparations,¹¹ and from vessels with simple endothelial injury,²¹ application of 100 nmol/L SNP to cells from rats exposed to nicotine caused no change in the current during a 10-minute or longer exposure (Figures 3B and 3C, open circles). Thus, chronic exposure to nicotine abolished the inhibitory effect of exogenous NO on Ca²⁺ channel currents.

View larger version (38K): [in this window] [in a new window]   Figure 3. Chronic nicotine blocks downregulation of Ca2+ channels by NO and cGMP. A and D, Current-voltage curves recorded during ramp pulse in LSA-SMCs from control animals before (a) and after (b) 100 nmol/L SNP (A) and before (a) and after (b) 100 µmol/L 8-bromo-cGMP (D). B and E, Current-voltage curves recorded during ramp pulse in LSA-SMCs from chronic nicotine animals before (a) and after (b) 100 nmol/L SNP (B) and before (a) and after (b) 100 µmol/L 8-bromo-cGMP (E). C and F, Effect of 100 nmol/L SNP (C) and 100 µmol/L 8-bromo-cGMP (8Br-cGMP) (F) on peak Ca2+ channel currents in LSA-SMCs from control (•) and chronic nicotine () animals; values (mean±SE) are plotted for 9 and 8 cells for SNP and for 6 and 7 cells for 8-bromo-cGMP, for control and chronic nicotine animals, respectively.

In smooth muscle, effects of NO are mediated by cGMP.¹¹ With cells from control animals, application of the membrane-permeable analogue, 8-bromo-cGMP, caused the expected downregulation of Ca²⁺ channel current (Figures 3D and 3F, closed circles). By contrast, in cells from chronic nicotine rats, 8-bromo-cGMP had no effect on Ca²⁺ channel currents (Figure 3E and 3F, open circles), corroborating the result obtained with NO donor.

NO and cGMP also upregulate BK channels.⁹ In cells from control rats, SNP (Figure 4A) and 8-bromo-cGMP caused a robust increase in outward current. In cells from chronic nicotine rats, both SNP (Figure 4B) and 8-bromo-cGMP (Figure 4C) increased the outward current in a manner indistinguishable from controls. Together, these findings with BK channels suggest that neither guanylate cyclase nor PKG was affected by nicotine, suggesting that the location for the block of NO- and cGMP-mediated downregulation of Ca²⁺ channels by nicotine was downstream of PKG.

View larger version (27K): [in this window] [in a new window]   Figure 4. Chronic nicotine does not block upregulation of Ca2+-activated K+ channels by NO and cGMP or downregulation of Ca2+ channels by tyrosine kinase inhibitor. A through C, Outward K+ channel currents in LSA-SMCs from control (A) and chronic nicotine (B and C) animals at baseline (Aa, Ba, and Ca) and after 100 nmol/L SNP (Ab and Bb) and 100 µmol/L 8-bromo-cGMP (Cb); shown are 200-ms depolarizing pulses to +80 mV from holding potential of 0 mV. D and E, Ca2+ channel current-voltage curves recorded during ramp pulse in LSA-SMCs from a control animal (D) and from a chronic nicotine animal (E), before (a) and after (b) 100 µmol/L tyrophostin (AG-18).

The data presented are consistent with the hypothesis that nicotine exerted complex effects by at least 2 mechanisms, as follows: (1) by apparent reduction in bioavailability of endogenous NO and (2) by interfering with NO signaling in smooth muscle. Exposure to nicotine also causes release of endothelin,²⁶ and in some smooth muscle preparations,²⁷ although not in cerebral vascular smooth muscle,²⁸ endothelin can upregulate Ca²⁺ channels. In LSA-SMCs, endothelin (0.1 to 100 nmol/L) caused no increase in current (11 cells). Also, to exclude possible direct stimulatory effects on Ca²⁺ channels or inhibitory effects on BK channels, we applied nicotine directly to cells from control animals. Application of nicotine (0.5 and 10 µmol/L) for 5 to 12 minutes caused no change in Ca²⁺ (9 cells) or BK (5 cells) channel current. Also, preexposure of cells to nicotine in vitro did not prevent subsequent normal downregulation of Ca²⁺ channel current (8 cells), indicating that nicotine was not acting as an NO scavenger. Finally, application of the tyrosine kinase inhibitor, tyrophostin (AG-18), resulted in significant and equivalent downregulation of Ca²⁺ channels in LSA-SMCs from both control (Figure 4D) and chronic nicotine (Figure 4E) rats, suggesting that loss of the response to NO/cGMP was not due to a nonspecific effect preventing phosphorylation or dephosphorylation of the channel. Together, these control experiments showed that altered channel availability observed with chronic nicotine was not due to either endothelin release or to direct effects of nicotine on Ca²⁺ or BK channels and that loss of NO signaling was not due to nonspecific effects on Ca²⁺ channels.

Finally, we sought to determine whether findings in isolated cells would predict effects in intact cerebral arterioles. We used LDF to measure changes in CBF and a pial window technique to measure pial arteriolar diameter. In control rats, infusion of the NO donor S-nitroso-N-acetylpencillamine (SNAP) caused a progressive increase in CBF (Figures 5Aa and 5B CONT) and in pial vessel diameter (Figure 5C CONT). By contrast, in chronic nicotine rats, the same protocol with SNAP resulted in significantly less vasodilation, as measured by both CBF and pial vessel diameter (Figures 5Ab, 5B NIC, and 5C NIC). The finding of a significant but incomplete block of NO effect in arterioles in vivo expands on previous observations of effects of nicotine in noncerebral vessels⁵ and corroborates the observation in isolated cells that chronic nicotine blocks NO effects involving Ca²⁺ but not BK channels, suggesting that both channels must be involved for full relaxation.

View larger version (25K): [in this window] [in a new window]   Figure 5. Chronic nicotine blunts cerebral vasodilation by NO. A, Increase in CBF measured by LDF in response to 10-minute topical infusion of 500 µmol/L NO donor, SNAP, in a control (a) and a chronic nicotine (b) animal; changes in blood flow, measured in tissue perfusion units (TPU), were computed after subtracting baseline values. B and C, Mean±SE for change in CBF (B) and pial vessel diameter (C) after a 10-minute topical infusion of SNAP in 6 control (CONT) and 8 chronic nicotine (NIC) animals; values significantly different by t test (P=0.003 [B] P=0.02 [C]).

	Discussion

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Chronic exposure to nicotine at concentrations comparable with those in humans who actively smoke¹⁵ resulted in 2 major types of effects on smooth muscle ion channels, as follows: (1) an alteration in ion channel availability, with Ca²⁺ channels upregulated and BK channels downregulated, and (2) a block in NO-mediated downregulation of Ca²⁺ channels but not of NO-mediated upregulation of BK channels.

An increase in availability of Ca²⁺ channels can arise by a variety of mechanisms, including a change in biophysical properties of the channel, a second messenger–mediated increase in open probability or open time of the channel, or an increase in expression of functional channels. The first of these was excluded by our data showing no effect of chronic nicotine on the biophysical or pharmacological properties of the Ca²⁺ channel. Although we have no data specifically excluding an increase in expression of Ca²⁺ channels, at present we favor an alteration in second messenger–mediated regulation as the most parsimonious explanation for both simultaneous upregulation of Ca²⁺ channels and downregulation of BK channels. Because NO normally downregulates Ca²⁺ channels^{11 12} and upregulates BK channels,^{9 10} the observed upregulation of Ca²⁺ channels and downregulation of BK channels mimics precisely the effects expected with an apparent decrease in bioavailability of endogenous NO. Indeed, we recently demonstrated that a decrease in bioavailability of endogenous NO, as well as block of endogenous NOS activity and oxidative endothelial injury, results in a significant increase in Ca²⁺ channel availability,²¹ similar to that observed here with chronic nicotine. Nicotine is known to cause desquamation and ultrastructural abnormalities in endothelium,^{29 30} and vasoconstrictive effects of nicotine have been postulated to be due to impaired release of NO,^{4 7} possibly as a result of oxidative stress.^{31 32} Thus, endothelial dysfunction, possibly initiated via endothelial nicotinic receptors,³³ would account well for the altered channel availability observed here. Additional work will be required to confirm by direct measurement the apparent reduction in bioavailability of endogenous NO in the smooth muscle layer of arterioles in vivo that is suggested by our data.

The second major effect of nicotine was the novel finding of a block of normal NO-mediated downregulation of Ca²⁺ channels. Our experiments not only established that nicotine blocks NO signaling involving Ca²⁺ channels, but they also helped localize the site of block in the signaling pathway. A critical experiment was the one showing maintenance of the effect of NO on BK channel upregulation. This finding essentially eliminates the initial steps in the signaling pathway, including guanylate cyclase, cGMP itself, or PKG, and points to a site downstream of PKG as the target of nicotine. An effect downstream of PKG suggests either that the channel itself is altered by nicotine, resulting in diminished sensitivity to PKG phosphorylation, or that the channel is not phosphorylated by PKG. Our experiments showing no effect of chronic nicotine on the biophysical or pharmacological properties of the Ca²⁺ channel provide no support for a hypothesis of an altered channel, and similarly our experiment showing no effect of chronic nicotine on downregulation with tyrosine kinase inhibitor argues that phosphorylation/dephosphorylation mechanisms directly involving the channel are not affected by nicotine. Alternatively, if the channel itself is not altered by nicotine, this would suggest that an intermediate regulatory phosphoprotein may be interposed between PKG and the channel and that this intermediate phosphoprotein is the target of nicotine. At present, the specific phosphoprotein target of PKG involved in NO-mediated downregulation of Ca²⁺ channels in smooth muscle is not known. In cardiac cells, Ca²⁺ channels are phosphorylated by PKG, resulting in a reduced open probability.³⁴ L-type Ca²⁺ channels are heteropentameric complexes, with the ₁ subunit subject to phosphorylation by PKG.³⁴ The molecular diversity of ₁ genes and the splice variants produced from these genes is extensive,^{35 36} however, raising the possibility that the ₁ subunit in cerebrovascular smooth muscle, unlike cardiac cells, may not be phosphorylated by PKG, or if it is, that phosphorylation does not cause a decrease in open probability. Further work will be required to clarify whether nicotine directly alters the Ca²⁺ channel to block PKG phosphorylation or whether nicotine blocks a putative intermediate regulatory phosphoprotein downstream of PKG that is involved in downregulating the channel.

Inhibition of Ca²⁺ influx is critical to relaxation of cerebrovascular smooth muscle. Thus, 2 distinct signaling mechanisms initiated by NO have evolved to inhibit Ca²⁺ influx. One of these is the signaling pathway that involves PKG-mediated phosphorylation of BK channels (Figure 6, lower branch). This pathway is voltage dependent, because the regulatory phosphoprotein, the BK channel, is coupled to its target, the Ca²⁺ channel, by virtue of the intrinsic voltage dependence of the Ca²⁺ channel. PKG-mediated phosphorylation of the BK channel increases its availability, serving to polarize the cell and thereby turn off voltage-dependent Ca²⁺ channels and decrease Ca²⁺ influx. This pathway has been extensively investigated,^{37 38} given the wide availability of K⁺ channel blockers.

View larger version (7K): [in this window] [in a new window]   Figure 6. NO inhibits L-type Ca2+ channel by 2 parallel mechanisms. Lower branch, Voltage-dependent, iberiotoxin-sensitive mechanism using BK channel as an intermediate regulatory phosphoprotein acting on the Ca2+ channel. Upper branch, Voltage-independent, nicotine-sensitive mechanism using unknown regulatory phosphoprotein (?) to act on the Ca2+ channel. PKG indicates cGMP-dependent protein kinase; Ca CHAN, Ca2+ channel; and , downregulation.

Less well understood is a second signaling pathway involving PKG-mediated downregulation of Ca²⁺ channels^{11 12} that is independent of K⁺ channels (Figure 6, upper branch). As noted above, the specific target of PKG in this branch remains to be identified, but our data suggest that it is this step involving action of an unidentified regulatory phosphoprotein on the Ca²⁺ channel that may be blocked either directly or indirectly by nicotine (Figure 6, upper branch). This pathway is voltage independent, given that the intrinsic voltage dependence of the Ca²⁺ channel is not involved in coupling the regulatory phosphoprotein to its target, the Ca²⁺ channel. The unique effect of nicotine reported here aids in delineating the specific contribution of the voltage-independent mechanism to the process of vasorelaxation, showing that approximately half of the vasodilatory response to NO in vivo was eliminated when the voltage-independent mechanism was blocked by nicotine. The present study using nicotine is the first to assess the important contribution of L-type Ca²⁺ channel inhibition, independent of BK channel–mediated voltage-dependent deactivation, in achieving NO-induced vasorelaxation.

The effects of chronic nicotine reported here provide both a mechanistic basis for abnormal vasorelaxation and a likely explanation for structural pathological effects. Increased availability of Ca²⁺ channels favors increased basal Ca²⁺ influx into smooth muscle cells that, if uncompensated, will lead to Ca²⁺-induced cell injury and cell death.³⁹ Tobacco smoke leads to numerous degenerative changes in cerebral vessels, including intimal hyperplasia, atherosclerosis, loss of smooth muscle cells, and aneurysm formation.⁴⁰ These pathological changes have been attributed to activation of matrix metalloproteinases, increased DNA synthesis, and cell proliferation.⁴¹ The data presented here indicate that nicotine-induced alteration of Ca²⁺ homeostasis may be responsible for smooth muscle cell toxicity and cell death.

	Acknowledgments

 
This work was supported by grants (to J.M.S.) from the National Heart, Lung, and Blood Institute (HL51932), the National Institute for Neurological Diseases and Stroke (NS39956), and the American Heart Association (a Burgher award). We thank Lioudmila Melnitchenko and Dr Jia Bi Yang for expert technical assistance.

	Footnotes

 
Original received September 11, 2000; revision received December 7, 2000; accepted December 8, 2000.

	References

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