Cyclic nucleotides and vasoconstrictor function: physiological and pathophysiological considerations

Pathophysiology 5 (1999) 233 – 245

Review article

Cyclic nucleotides and vasoconstrictor function: physiological and pathophysiological considerations Mark S. Taylor, A. Marie McMahon, Jason D. Gardner, Joseph N. Benoit * Department of Physiology, Uni6ersity of South Alabama, College of Medicine, MSB 3024, Mobile, AL 36688, USA Accepted 15 September 1998

Abstract The role of cyclic nucleotides as modulators of vascular smooth muscle tone has been widely studied. Yet the cellular mechanisms whereby cAMP and cGMP promote vascular relaxation remain highly controversial. The purpose of this review is to summarize a large and expanding literature with particular emphasis on the cellular mechanisms of cAMP- and cGMP-induced relaxation of vascular smooth muscle. The review addresses the following topics: regulation of vascular tone, mechanisms of cAMP- and cGMP-mediated vascular smooth muscle relaxation, modulation of [Ca2 + ]i by cAMP and cGMP, effects of cAMP and cGMP on the Ca2 + sensitivity of the contractile apparatus, and pathophysiology of the resistance vasculature in chronic portal hypertension with particular emphasis on cAMP and cGMP dependent pathways. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vascular smooth muscle; cAMP; cGMP; Phosphodiesterases; Calcium; Portal hypertension

1. Introduction Numerous studies in recent years have provided evidence that vasoactive agents modulate cyclic nucleotide levels in vascular smooth muscle cells. Although the role of adenosine 3%, 5%-cyclic monophosphate (cAMP) and guanosine 3%, 5%-cyclic monophosphate (cGMP) as cellular mediators of vasodilation has been established, only recently have the cellular mechanisms of these second messenger systems become evident. From these studies it is now apparent that cyclic nucleotides can modulate vascular smooth muscle tension by altering cytosolic calcium levels as well as by directly modulating the contractile machinery of the cell. In spite of emerging data in normal vascular smooth muscle, less is known of the potential role that cyclic nucleotides play as mediators of vascular dysfunction in pathologic * Corresponding author. Tel.: + 1 334 460 6833; fax: +1 334 460 6464; e-mail: [email protected]

states. The purpose of this review is to summarize current mechanisms of how cyclic nucleotides modulate vascular contractile behavior, as well as to summarize a developing literature that links vascular contractile dysfunction in chronic portal hypertension to cAMP or cGMP dependent events.

2. Regulation of vascular tone There is a wealth of evidence to support the contention that regulation of vascular smooth muscle tone and ultimately vascular resistance is dependent on a tightly coupled balance between vasoconstrictor and vasodilator influences. Factors tending to shift the balance towards constriction increase vascular resistance, while factors that shift the balance toward dilation decrease vascular resistance. Studies in recent years have more clearly defined the cellular pathways associated with constriction and relaxation of vascular

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Fig. 1. Key cellular events associated with pharmacomechanical and electromechanical coupling. Note that L-type Ca2 + channels (L) can be activated by either pathway. Dotted lines indicate inhibitory effects. (Abbreviations: SOC, store-operated Ca2 + channels; ROC, receptor-operated Ca2 + channels; R, G-protein coupled receptor; PLA2, phospholipase A2; PLC, phospholipase C; PC, phosphatidylcholine; AA, arachidonic acid; PIP2, phosphatidylinsitol 4,5-bisphosphate; DAG, 1,2-diacylglycerol; PKC, protein kinase C; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; RyR, ryanodine receptor; Cam, calmodulin; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; SERCA, sarcoplasmic/ endoplasmic reticulum calcium-ATPase; CaP, calponin; CaD, caldesmon; A, actin; M, myosin).

smooth muscle. In spite of a growing literature on cellular regulatory pathways in vascular smooth muscle, many investigators continue to classify physiological and pharmacological modulators of vascular resistance according to their global effects (i.e. dilators or constrictors) and not their cellular mechanisms of action. In the sections that follow, key cellular events associated with vasoconstriction and vasodilation are described.

2.1. Mechanisms of 6ascular smooth muscle contraction Contractile stimuli produce an increase in intracellular calcium, phosphorylation of myosin and cross bridge formation. The dependence of smooth muscle contraction on calcium (Ca2 + ) was first determined by Filo et al. [1], who demonstrated that a critical concentration range of Ca2 + (180 – 1000 nM) was needed to

evoke contraction of permeabilized vascular smooth muscle. Myosin phosphorylation, a necessary event for smooth muscle contraction, was later shown to be a Ca2 + -dependent process [2]. Dabrowska et al. [3] consequently identified myosin light chain kinase (MLCK) in vascular smooth muscle. The calcium dependent modulator protein, calmodulin, has been shown to activate MLCK, which phosphorylates the regulatory light chains of myosin. This phosphorylation increases the affinity of myosin filaments for binding sites on the actin filaments. Based upon these findings, a general model of smooth muscle contraction has been proposed (Fig. 1). Stimulation of smooth muscle cells causes an increase in cytosolic Ca2 + concentration ([Ca2 + ]i) [125]. Calcium ions form complexes with calmodulin (four Ca2 + ions, one calmodulin molecule) which binds to and activates MLCK [4,5]. Active MLCK transfers the terminal phosphates of ATP to regulatory light

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chains of myosin, thereby altering the affinity of myosin for actin. The terminal phosphates are transferred to serine-19 on each of the two 20 kDa myosin light chain subunits (also called LC20). Binding of actin then stimulates myosin ATP-ase, resulting in myosin cross-bridge cycling along actin filaments and muscle contraction [6]. Relaxation of vascular smooth muscle involves the reversal of the aforementioned processes. Reductions in cytosolic calcium result from removal of the contractile stimulus as well as the sequestration and extrusion of Ca2 + by the sarcoplasmic reticulum (SR), and plasma membrane Ca2 + -ATPases, respectively [7]. Myosin light chain phosphatase (MLCP) dephosphorylates myosin which ultimately leads to a reduced number of actin-myosin crossbridges [8]. Alternatively, the complex of actin and phosphorylated myosin may spontaneously dephosphorylate [9]. It is not known whether this step involves specific phosphatases or if it can be physiologically regulated. Nonetheless, dissociation of the remaining actin-myosin complex is relatively slow, allowing for the maintenance of contraction at a reduced rate of energy expenditure [10]. The existence of this ‘latch state’ has been difficult to demonstrate experimentally. However it remains the only acceptable model that can explain the dissociation between tension and energy expenditure during tonic isometric contraction of smooth muscle.

2.2. Excitation contraction coupling in 6ascular smooth muscle: electromechanical and pharmacomechanical e6ents In order to begin to understand the cellular mechanism whereby pathological states can lead to altered vasoregulatory function, one must first consider the means by which vascular smooth muscle generates force. Two primary mechanisms of vascular smooth muscle contraction, electromechanical and pharmacomechanical, have been described. Electromechanical coupling begins with membrane depolarization via the gating of ion channels (e.g. K + , Cl − , Ca2 + ). As a result of the altered electrochemical gradient, voltagegated Ca2 + channels open (L- and T-channels), allowing the diffusion of Ca2 + down its concentration gradient and into the cell [11,12]. Under these conditions, the primary trigger for force development appears to be calcium availability. Pharmacomechanical coupling, does not require cell depolarization, but involves the binding of a ligand (e.g. norepinephrine, vasopressin, angiotensin II) to a G-protein coupled membrane receptor [13,14]. Membrane associated Gproteins are heterotrimers comprised of a, b, and g subunits. Upon activation, the a-subunit dissociates from the bg-subunits to trigger a cascade of events. Differences in Ga-subtypes confer some degree of specificity to the G-protein response. Three key a-subtypes

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are involved in G-protein coupled responses of vascular smooth muscle. The Gas and Gai subtypes activate, and inhibit adenylyl cyclase, respectively. The physiological implications of altered cAMP levels will be discussed in a subsequent section. The aq-subunit of G-proteins induces vasoconstriction primarily through the activation of cellular phospholipases. For constrictors such as a-adrenergic agonists, the b-isoform of phospholipaseC (PLC-b) is stimulated. This enzyme hydrolyzes membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2), yielding inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) [14]. The targets of each of these cleavage products differ. DAG activates protein kinase-C (PKC) which is known to increase ionic conductance through transmembrane channels (L-type Ca2 + and K + channels) [15]. PKC is also believed to modulate the calcium sensitivity of the contractile apparatus [16]. IP3 binds receptors on the SR membrane causing the release of Ca2 + into the cytoplasm [17–19]. Furthermore, muscarinic or a1-adrenergic agonists can bind to G-protein coupled receptors and activate phospholipase A2 (PLA2) causing generation of arachidonic acid. Arachidonic acid inhibits MLCP thereby increasing calcium sensitivity [8]. In addition to the well-defined L-type channels, the smooth muscle cell membrane also contains other types of Ca2 + channels. Release of Ca2 + from the SR has been shown to induce Ca2 + influx through store-operated channels (SOC) on the plasma membrane. This mechanism allows for the refilling of depleted internal Ca2 + stores (hence the term capacitative Ca2 + entry) and allows for additional increases in cytoslic Ca2 + concentration [20]. The signaling process whereby the SOC detects a depletion of SR Ca2 + stores is not fully understood, although mechanisms involving mechanical coupling or a diffusible messenger are postulated. Recent preliminary studies from our laboratory have examined the contribution of capacitative Ca2 + entry in the Ca2 + -response of aortic vascular smooth muscle cells to vasopressin stimuli. Although we were able to demonstrate capacitative Ca2 + entry following depletion of SR Ca2 + , we were not able to demonstrate any role for capacitative entry in cells stimulated with vasopressin under normal conditions [21]. To this end, the role of capacitative Ca2 + entry remains unclear. Theories suggest that there may be a ‘preferred pathway’ where Ca2 + influx through the SOC preferentially enters the SR from the extracellular space such that the Ca2 + is unavailable to the contractile machinery. Another source of calcium influx is the receptor operated Ca2 + channel (ROC). Binding of a vasoconstrictor to its receptor may elicit Ca2 + influx or cause depolarization and subsequent activation of L-type Ca2 + channels via mechanisms that do not involve G-proteins. Studies focusing on the functional role of ROCs have found that these channels do contribute to calcium

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influx in cultured aortic cells [22], but not in mesenteric artery smooth muscle [23]. T-type channels, are voltagegated channels on cell membranes which open transiently in response to cell depolarization. However, unlike the L-type channels, T-type channels do not appear to contribute substantially to the regulation of intracellular calcium levels in vascular smooth muscle [15]. The route of Ca2 + entry and its relative contribution to constriction appear to be agonist-dependent. Diacylglycerol activates PKC, which may enhance contraction in various ways. PKC may phosphorylate and open L-type Ca2 + channels, eliciting an increase in Ca2 + influx. PKC may also increase Ca2 + influx through L-type Ca2 + channels indirectly by phosphorylating and inhibiting membrane potassium channels, allowing for cell depolarization [15,24 – 27]. Additionally, PKC has been suggested to enhance Ca2 + sensitivity in smooth muscle, possibly through inhibition of MLCP or phosphorylation of microfilament regulatory proteins such as caldesmon and calponin [28 – 30]. Although many of its actions remain unclear, PKC appears to play an integral role in vascular smooth muscle contraction [31,32]. In both electromechanical and pharmacomechanical coupling, the resultant increase in [Ca2 + ]i may cause further Ca2 + release through stimulation of ryanodinesensitive receptors located on SR membranes [35,36]. This Ca2 + -induced Ca2 + release (CICR) has been implicated in a number of complex intracellular Ca2 + signaling events such as Ca2 + oscillations and waves [37,38]. The events of pharmacomechanical and electromechanical contraction of vascular smooth muscle are summarized in Fig. 1.

2.3. Other contributors to smooth muscle contraction In addition to the events considered above, a number of other factors may contribute to smooth muscle contraction. One such event is the opening of stretch-activated Ca2 + channels that are mechanosensitive cation channels believed to be important for normal myogenic control of vascular tone. Alteration in the activity of the Na2 + –Ca2 + exchanger has also been suggested to affect vascular reactivity as well as stimulation of receptor tyrosine kinases [33,34]. Although the physiological significance of many of these factors has not been resolved, their combined role in typical vasoconstrictor responses is thought to be minimal.

3. Mechanisms of vascular smooth muscle relaxation Removal of the contractile stimulus is the first step towards smooth muscle relaxation. The primary mechanism of smooth muscle relaxation is reduction of free

cytosolic calcium concentration through SR sequestration and plasmalemmal pump activity. Reduction of calcium leads to the dissociation of calmodulin from MLCK. Myosin is then dephosphorylated by MLCP which minimizes actin/myosin interactions, thereby disengaging the contractile mechanism. However, certain ligands (e.g. b-adrenergic agonists, glucagon, prostacyclin) can cause receptor-mediated active relaxation of vascular smooth muscle. Other substances such as nitric oxide cause active vasorelaxation by receptor-independent pathways. Receptor-mediated relaxation is most often associated with elevations in cyclic nucleotides [39,40]. The formation of cyclic nucleotides and their role in the cellular mechanisms of relaxation and calcium regulation will be discussed in the following sections.

4. Mechanisms of cAMP-mediated vascular smooth muscle relaxation Various vasodilatory stimuli have also been investigated in recent years. These factors are thought to relax smooth muscle by opposing the vasoconstrictor mechanisms described above (see Fig. 2). Most often, vasodilation is associated with the elevation of cyclic nucleotides, cAMP and cGMP, in vascular smooth muscle cells [39–42]. Cyclic AMP is generated through the binding of certain ligands (e.g. b-agonists, prostaglandins, glucagon) to membrane receptors which triggers the dissociation of the heterotrimeric Gs protein [43]. The Gas subunit activates the membrane-bound adenylyl cyclase, which catalyzes formation of cAMP from ATP [44]. Cellular functions of cAMP are manifested through the activation of cAMP-dependent protein kinase (PKA) [45]. Saturation of the regulatory subunits with cAMP causes dissociation and activation of the two catalytic subunits of this tetrameric kinase. The catalytic subunits of PKA catalyze the phosphorylation of various cellular targets, altering their activity. Cyclic AMP is degraded by cellular phosphodiesterase (PDE) enzymes, producing the inactive metabolite 5% AMP. In vascular smooth muscle, these PDEs are typically comprised of the cAMP-specific isoform PDE4 and the cGMP-inhibitible isoform PDE3. Cyclic AMP is postulated to inhibit vasoconstriction by two distinct mechanisms, (1) decreasing VSM cell Ca2 + concentrations and (2) by reducing Ca2 + -sensitivity of the contractile apparatus. McDaniel et al. [46] found that elevating cAMP with the adenylyl cyclase activator forskolin reduced Ca2 + concentrations in swine arterial smooth muscle as measured with aequorin. A number of investigators have reported similar effects on myoplasmic Ca2 + concentration during periods of cAMP elevation [7,15,31,39,47–49]. Since smooth muscle contraction is calcium dependent, one

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Fig. 2. Schematic representation of cyclic nucleotide mechanisms of action in vascular smooth muscle cells. The extracellular signals (agonists), plasma membrane receptors and intracellular components involved in cAMP and cGMP mediated events are shown. Arrows blocked by a positive sign ( + ) indicate a stimulatory effect, whereas those blocked by a negative sign ( − ), indicate inhibition. Important elements for contractile regulation include: inositol 1,4,5-trisphosphate receptor (IP3R), L-type Ca2 + channel (L), plasma membrane K + channel (K), phospholamban (Plb), Ca2 + -ATPase pump (SERCA) and MLCK. Important elements for regulation of relaxation include: Nitric oxide (NO), Atrial natriuretic peptides (ANP), adenylyl cyclase (AC), membrane-bound guanylyl cyclase (GCm), cystolic guanylyl cyclase (GCc), guanosine triphosphate (GTP), 3%, 5%-cyclic guanosine monophosphate (cGMP), adenosine triphosphate (ATP), 3%, 5%-cyclic adenosine monophosphate (cAMP), cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), myosin light chain phosphatase (MLCP), myosin light chain kinase (MLCK) and phosphodiesterases 3 (PDE3), 4 (PDE4) and 5 (PDE5).

may conclude that cAMP relaxes smooth muscle by lowering calcium availability. However, evidence indicating that cAMP substantially alters the association between [Ca2 + ]i and tension also exists. Recent research efforts have focused on identifying pathways of Ca2 + mobilization that are regulated by cyclic nucleotides as well as elucidating the mechanisms responsible for the reduction of Ca2 + -sensitivity.

5. Cyclic AMP modulation of [Ca2 + ]i

5.1. Effects of cAMP on Ca 2 + influx A number of investigators have reported cAMP-mediated alteration of Ca2 + influx from the extracellular

compartment. Unlike skeletal and cardiac muscle, where cAMP elevation increases Ca2 + entry through PKA phosphorylation of voltage sensitive L-type Ca2 + channels [11,50], cAMP inhibits inward Ca2 + current through membrane Ca2 + channels in vascular smooth muscle. Dibutyryl and 8-bromo derivatives of cAMP have been shown by Xiong and Sperelakis [15] to substantially reduce L-type Ca2 + current (ICa(L)) in freshly isolated and cultured VSM cells, with the peak effect occurring after 10 min. These effects were prevented by administration of the non-specific protein kinase inhibitor H-7. Introduction of the catalytic subunit of PKA in patch clamp studies significantly reduced Ica(L), supporting the hypothesis of direct channel phosphorylation [15]. Similar effects on Ca2 + influx were seen with cGMP elevation and cGMP-dependent

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protein kinase (PKG) activation [39]. In spite of these studies, no direct biochemical evidence supporting Ltype Ca2 + channel phosphorylation has been reported. It remains unclear how PKA inhibits activity of L-type Ca2 + channels in vascular smooth muscle and stimulates L-type Ca2 + channels in skeletal and cardiac muscle although the existence of cell-specific regulatory proteins seems most likely. It has been proposed that cyclic nucleotide reduction of voltage dependent Ca2 + influx and associated vascular smooth muscle relaxation may result from regulation of membrane K + channels. Under such conditions, cyclic AMP would increase K + efflux through these channels, thereby hyperpolarizing the cell and limiting Ca2 + influx through the voltage sensitive L-type channels. Four types of K + channels have been identified in vascular smooth muscle plasma membranes [51]. The voltage-gated K + channel (Kv) regulates smooth muscle membrane potential in response to depolarizing stimuli. Calcium-activated K + channels (KCa) are activated by cytosolic Ca2 + as well as changes in membrane potential. ATP-sensitive K + channels (KATP) are sensitive to cell metabolic status (i.e. open in response to low ATP) and a number of other intracellular signals. The inward rectifying potassium channel (KIR) is the least characterized of these channels, but is known to open under hyperpolarizing conditions. Recent evidence indicates that some of these channels are regulated by vasodilator stimuli. Calcitonin gene related peptide (CGRP) has been found to hyperpolarize and relax smooth muscle in gallbladder, cerebral vessels and mesenteric vessels. Muscle relaxation by CGRP is mediated via cAMP elevation and subsequent PKA stimulation of KATP channels [51,52]. Subsequent studies have also revealed cAMP/PKA dependent regulation of KATP channels in cardiac and smooth muscle [27]. However, other vasodilators (e.g. b-agonists and adenosine) may activate KATP channels through a Gprotein pathway that does not directly involve cyclic nucleotides or their protein kinases [53]. In view of the known cAMP and KATP channel dependency of many vasodilators, careful interpretation is encouraged when assessing the role of cyclic nucleotides in agonist-induced responses. Voltage sensitive K + channels (Kv) are important in the regulation of vascular smooth muscle membrane potential, particularly the pressure-dependent (myogenic) responses in small arteries. Knot and Nelson [54] reported that the Kv channel blocker 4-aminopyridine enhanced myogenic responses in small cerebral arteries and that this effect was prevented by treatment with the Ca2 + channel blocker nisoldipine. Thus it appears that the Kv channel is involved in feedback regulation of Ca2 + influx that normally occurs with vessel stretch

and depolarization. A role for cAMP as a modulator of Kv channels has been provided in whole-cell patch clamp experiments using dispersed portal vein and coronary artery smooth muscle cells. In these studies, Cole et al. [26] revealed that Kv channels may be stimulated by PKA-dependent phosphorylation. Furthermore, it was shown that either isoproterenol or forskolin induction of Kv current was prevented by the endogenous PKA inhibitor PKI. These data indicate that cAMP may alter the sensitivity of vascular myogenic responses to changes in intramural pressure. Calcium activated K + channels (KCa) may also be regulated by vasodilators. Isoproterenol, forskolin, and dibutyryl-cAMP have been shown to activate KCa current in aortic myocytes and in cerebral and coronary arteries [51,55]. This KCa channel activation has been linked to PKA- and PKG-mediated channel phosphorylation as well as a protein kinase independent receptor-stimulated G-protein pathway [56,57]. These channels are believed to mediate spontaneous transient outward currents (STOCs) that occur across vascular smooth muscle cell membranes. STOCs appear to be triggered by periodic release of Ca2 + from subsarcolemmal ryanodine sensitive Ca2 + stores (calcium sparks), or from influx of Ca2 + ; the result of which is the activation and opening of membrane KCa channels [58,59]. This outward K + current hyperpolarizes the cell, reduces Ca2 + influx through L-type Ca2 + channels, and promotes vasorelaxation [60]. Inwardly rectifying K + channels (KIR) may be crucial for the maintenance of membrane potential in some small resistance arteries (coronary, cerebral and mesenteric), but their physiological role remains unclear [57]. A unique characteristic of these channels is that they may be activated by membrane hyperpolarization, yielding a net inward K + flux. They may also be activated by low concentrations of extracellular K + (1–15 mM). Under normal physiological conditions, KIR channels are thought to predominantly render outward K + currents, and may be involved in the K + -mediated vasodilation observed in cerebral [51], and intestinal vessels. Possible regulation of these channels by vasoactive substances and cyclic nucleotide-dependent processes has not yet been investigated. In spite of the wealth of data documenting cyclic nucleotide modulation of potassium channel activity, functional data supporting such a link is scant. Recent studies from our laboratory indicate that the inhibition of norepinephrine constriction by the PDE3 inhibitor milrinone is conserved regardless of K + channel function. This finding suggests a minimal contribution of K + channel regulation to the overall cAMP-mediated inhibition of VSM contraction [61]. Chloride channels may also be gated by cAMP, resulting in an increased inward Cl − current and hy-

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perpolarization. Zhao et al. [62] have recently demonstrated at least a partial dependence of forskolin-induced relaxation on transmembrane Cl − gradient in K + -constricted pulmonary artery. The potential physiological relevance of such regulation requires more investigation.

5.2. Effects of cAMP on Ca 2 + release and uptake Cyclic AMP regulation of [Ca2 + ]i may occur through an alteration in Ca2 + release from internal stores. PKA-dependent phosphorylation of IP3 receptors has been observed in vitro [63]. The consequences of this phosphorylation, however, are difficult to interpret. In cerebellar neuronal membranes, cAMP elevation decreases the potency of IP3 at the IP3 receptor but increases the accumulation of Ca2 + in the endoplasmic reticulum (ER). This causes an overall augmentation of absolute Ca2 + release [17]. In hepatocytes, cAMP tends to increase the sensitivity of ER stores to IP3 [64]. Generation of cAMP in tracheal smooth muscle has been shown to inhibit IP3 binding to SR IP3 receptors [65]. The effects of cAMP elevation on Ca2 + release in vascular smooth muscle have not been elucidated. Another potential site of regulation that has been a focus of recent study is the Ca2 + -ATPase. Kimura et al. [47] revealed that cholecystokinin (CCK) increased cAMP and lowered [Ca2 + ]i in hog bile duct. This response was linked to a PKA-mediated increase in SR sequestration, a finding consistent with the cAMP-stimulated increase in the sarcoplasmic/endoplasmic reticulum Ca2 + -ATPase (SERCA). Stimulation of SERCA is known to occur in cardiac muscle via phosphorylation of the regulatory protein phospholamban [66]. Similar regulation is believed to occur in vascular smooth muscle [67]. Normally, phospholamban in its dephosphorylated state disinhibits Ca2 + -ATPase activity. Phosphorylation inhibits phospholamban thereby increasing Ca2 + pump activity, allowing for the rapid sequestration of Ca2 + into the SR and a consequential decrease in [Ca2 + ]i. Protein kinase G-dependent phosphorylation of phospholamban was found to occur at the same site as PKA-mediated phosphorylation [68]. Lincoln et al. [49] have since proposed that PKG is the principle mediator of both cAMP- and cGMP-dependent relaxation of vascular smooth muscle. In their studies, cAMP elevation by way of forskolin did not affect AVP-induced Ca2 + mobilization in PKG deficient cells. Returning active PKG to these cells restored the cAMP mediated attenuation of AVP-induced Ca2 + . These investigators suggest that PKG-dependent phosphorylation of phospholamban at the SR membrane is a major mechanism of cyclic nucleotide-mediated relaxation.

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6. Effects of cAMP on Ca2 + -sensitivity As previously mentioned, cyclic nucleotides may inhibit smooth muscle contraction not only by limiting [Ca2 + ]i but also by interfering with contractile mechanisms downstream of Ca2 + mobilization (i.e. calcium sensitivity). Nishimura and van Breemen [69] found that cAMP attenuated constriction of a-toxin permeabilized rat mesenteric artery at constant calcium concentrations. This finding is indicative of a down regulation of myofilament Ca2 + -sensitivity. Alterations in Ca2 + sensitivity are thought to derive primarily from changes in MLC phosphorylation. It is well known that PKA-mediated phosphorylation of MLCK at serine 815 (site A) can reduce the affinity of MLCK for the Ca2 + –calmodulin complex, resulting in reduced smooth muscle tension generation [70]. However, evidence of substantial PKA-mediated phosphorylation under physiological conditions is lacking. In cases where site A is phosphorylated, CaM kinase II rather than PKA is most often responsible [71]. CaM kinase II phosphorylates site A to desensitize the apparatus in the absence of elevated cAMP. Recent data from Kotlikoff and Kamm [56] suggests that b-adrenergic receptor stimulation in airway smooth muscle desensitizes contractile elements to Ca2 + , possibly through activation of MLCP. Through this mechanism, myosin phosphorylation would proceed normally, but the activated phosphatase would quickly dephosphorylate the myosin head and prevent crossbridge cycling. A number of investigators have supported the idea of cAMPmediated stimulation of MLCP activity [8,69,72–74]. However, little evidence has been found of cAMP-dependent phosphorylation and activation of MLCP. Limited in vitro data indicates that PKA actually phosphorylates the large target subunit of MLCP, causing it to alternate between a membrane-bound, inactive dephosphorylated state and a cytosolic or cytoskeletalassociated, active phosphorylated state [73]. Recently, Wu et al. [74] proposed that the cyclic nucleotide-mediated Ca2 + -desensitization may result from PKA or PKG-dependent phosphorylation of the regulatory protein telokin. This kinase-related protein associates with the regulatory light chain (MLC20) and accelerates its dephosphorylation, possibly through facilitation of MLCP activity. This activity is enhanced by cyclic nucleotide-dependent protein kinase stimulation. Additional research is needed to define the physiological role of telokin in VSM contraction. Recent data from our laboratory supports the contention that cAMP alters the Ca2 + -sensitivity of the contractile elements. Using a preparation that allows for simultaneous measurement of vascular smooth muscle calcium and tension, we were able to observe cAMP dependent reductions in contractile tension without concomitant reductions in [Ca2 + ]i. These findings sug-

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gest that alterations in Ca2 + -sensitivity of the contractile apparatus account for a significant portion of cAMP-induced inhibition of Ca2 + -dependent tension development (Taylor, Gardner, and Benoit, unpublished observation).

7. Mechanisms of cGMP-mediated vascular smooth muscle relaxation The importance of cGMP in smooth muscle physiology has gained considerable attention in recent years. Early work in this field by Katsuki et al. [75] demonstrated that certain nitrogen compounds, such as nitric oxide (NO), stimulated guanylyl cyclase. Subsequent studies have shown that when smooth muscle preparations were exposed to inorganic nitrogen compounds or classic vasodilators (e.g. sodium nitroprusside or nitroglycerine), vasorelaxation was accompanied by increased cGMP levels [76,77]. Currently, cGMP is accepted as the intracellular mediator responsible for vascular relaxation caused by agents such as nitric oxide, inorganic nitrogen compounds and atrial natriuretic peptides (ANP). In the following section, several of the proposed mechanisms of action of cGMP on vascular smooth muscle contraction and relaxation will be discussed. The production of cGMP is stimulated by the enzymatic activity of guanylyl cyclase on guanosine triphosphate (GTP). There are two types of guanylyl cyclase in vascular smooth muscle cells. One is the particulate enzyme, which is actually a transmembrane spanning receptor that contains guanylyl cyclase within the intracellular domain [77]. The other form is free in the cytoplasm and referred to as soluble guanylyl cyclase [68]. Particulate guanylyl cyclase binds ANPs, whereas soluble guanylyl cyclase is stimulated by NO [8]. The most likely endogenous NO source for vascular smooth muscle is neighboring endothelial cells [78]. Exogenous donors of NO include sodium nitroprusside (SNP), sodium nitrite and nitroglycerine. A direct relationship between NO and cGMP has been demonstrated. Treatment of hamster thoracic aorta with SNP increased cGMP in endothelium-intact and endothelium-denuded vessel preparations by similar amounts [78]. It was concluded in this study that NO stimulated the production of cGMP in vascular smooth muscle. The physiological effects of cGMP in cells are achieved by its interactions with intracellular proteins. There are three main types of target proteins for cGMP, (1) PKGs, (2) cGMP-regulated ion channels, and (3) cGMP-phosphodiesterases [42]. The latter degrade cGMP. Thus, cyclic GMP can alter cell function through protein phosphorylation or through mechanisms not directly related to protein phosphorylation [42].

The mechanisms of action of cGMP are analogous in some respects to cAMP in that cGMP has been found to act predominantly through the activation of PKG. Unlike PKA, PKG is made up of two identical subunits with regulatory and catalytic domains within the same peptide [39,42]. Binding of cGMP induces a conformational change in the kinase, exposing the catalytic sites. The activated catalytic subunits of the kinase serve to phosphorylate cellular proteins, enzymes and ion channels which may lead to reduced free calcium in the sarcoplasm.

8. Cyclic GMP modulation of [Ca2 + ]i

8.1. Effects of cGMP on Ca 2 + influx It has been well established that NO can stimulate cGMP production via guanylyl cyclase [78]. Millette and Lamontange [79] showed that there was an endothelium-dependent and NO-mediated attenuation of vasopressin induced contractile tension in rat aortic rings. There may also be a link between NO, cGMP and K + channel-mediated hyperpolarization in vascular smooth muscle. Khan et al. [80] showed that NO, nitroglycerine and acetylcholine produced dose-dependent vasorelaxation in precontracted isolated rabbit mesenteric arteries. Blockade of the calcium-activated K + channels (KCa) by charybdotoxin or iberiotoxin significantly inhibited relaxation thereby implicating KCa channels in cGMP-mediated vasorelaxation. Whether or not the activation of K + channels was directly mediated by cGMP or NO remains to be determined. In a study of membrane potential by Murphy and Brayden [81], it was shown that NO, released by 3-morpholinosydnonimine, reversibly hyperpolarized rabbit mesenteric arteries. The hyperpolarization was blocked by glibenclamide (a KATP channel blocker) but not by blockers of KV or KIR channels. These investigators suggested that NO hyperpolarizes vascular smooth muscle by activating KATP channels, with the accumulation of cGMP as an intermediate step. This information, along with the KCa channel data, lends weight to the argument that cyclic nucleotides act to modulate K + channels. To this end, endothelial derived NO and therapeutic agents such as nitrates predominately cause smooth muscle relaxation via cGMP.

8.2. Effects of cGMP on Ca 2 + release and uptake There are several possible mechanisms by which cyclic nucleotides modulate the concentration of intracellular Ca2 + ions and hence vascular smooth muscle tone. Like cAMP, there is evidence to suggest that phosphorylation of the regulatory protein phospholam-

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ban allows for activation of the Ca2 + pump on the SR, which in turn reduces [Ca2 + ]i. Dephosphorylated and phosphorylated phospholamban inhibits, and stimulates Ca2 + -ATPase on the SR, respectively. Although phospholamban phosphorylation has been traditionally attributed to cAMP pathways, similar phosphorylation and inhibition of phospholamban has been reported for [32P]-labeled aortic smooth muscle following cGMP elevation [68]. This PKG-dependent phosphorylation of phospholamban was found to occur at the same site as PKA-mediated phosphorylation. Cyclic GMP also enhances SR sequestration of Ca2 + ions in [45Ca]-loaded, saponin-skinned primary cultures of rat aortic myocytes [82]. As discussed previously, Lincoln et al. [49] proposed that PKG-dependent phosphorylation of phospholamban at the SR membrane is a major mechanism of cyclic nucleotide-mediated relaxation. It is also possible that Ca2 + -ATPase activity at the plasma membrane may be regulated by cGMP. Elevation of cGMP with SNP, ANP or 8-Br-cGMP accelerates Na + -independent Ca2 + efflux from vascular smooth muscle cells [48]. The calcium efflux that is not dependent on the relative external Na + concentration is attributable to membrane Ca2 + -ATPase activity and not Na + –Ca2 + exchange. These findings suggest that cGMP may mediate Ca2 + efflux by phosphorylation and activation of Ca2 + -ATPase on the plasma membrane. However, since PKG has been localized predominately to the SR in close association with phospholamban, one may argue that the more physiological role of PKG occurs through enhancement of calcium sequestration by the sarcoplasmic reticulum rather than via augmentation of Ca2 + -extrusion [66].

9. Effects of cGMP on Ca2 + -sensitivity Currently, there is little information on the effects of cGMP downstream of Ca2 + in vascular smooth muscle, that is, at sites that do not directly involve intracellular Ca2 + ion mobilization. However, data from intestinal smooth muscle has yielded important information and may also lead to analogous findings in vascular smooth muscle. Wu et al. [74] have recently found that telokin accelerated dephosphorylation of the myosin light chain (MLC) by enhancing MLCP activity in rabbit ileum smooth muscle. Telokin phosphorylation was enhanced not only by cAMP elevation but also by the action of the cGMP analog, 8-Br-cGMP [74]. It was concluded that PKG as well as PKA-dependent phosphorylation of endogenous telokin contributed to Ca2 + -desensitization, an event believed to be mediated by activation of MLCP. This data may have important implications on the control of vascular smooth muscle cell regulation. Recent data from our laboratory indicate that a moderate reduction in Ca2 +

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ion mobilization (28%) in cultured rat aortic cells occurs after pretreatment with the NO donor spermine/ NO [83]. However, the application of spermine/NO to aortic ring preparations causes a much larger reduction in vessel tension (\ 90%).

10. Pathophysiology of the resistance vasculature in chronic portal hypertension: cAMP and cGMP dependent pathways The response of the gastrointestinal circulation to cirrhosis and portal hypertension has been intensely studied in past years [84,85]. One of the most controversial and most studied consequences of portal hypertension is the origin of the hyperdynamic circulation. The term ‘hyperdynamic circulation’, is used to describe the reduced peripheral vascular resistance and elevated cardiac output that accompany the portal hypertensive condition. Early studies from our laboratory invoked a vasodilator shunting hypothesis to explain the gastrointestinal hemodynamic consequences of portal hypertension [86]. According to this mechanism, obstruction of the portal venous territory leads to the development of collateral vessels that divert portal blood away from the liver and into the systemic circulation. The resultant shunt flow limits metabolic clearance of vasoactive agents by the liver and leads to elevations of these substances in the arterial blood. Increases in vasoactive agents mediate relaxation of the peripheral vasculature. Since the inception of this hypothesis, many substances have been postulated to serve as humoral vasodilators in portal hypertension. To date, the literature supports glucagon [86–88], and prostacyclin [89,90], as prime mediators. The role of endothelial derived NO in the hyperdynamic circulation is far more controversial [91–96]. Nonetheless, the literature contains sufficient evidence to invoke cAMP and possibly cGMP pathways in the origin of the hyperdynamic circulation. Perhaps the most intriguing vascular consequence of portal hypertensive conditions is a decrease in the ability of blood vessels to respond to vasoconstrictor stimuli. Reduced responsiveness to a variety of agonists, including norepinephrine [97–101], vasopressin [102], and angiotensin II [90,103], have been observed in portal hypertensive states. Early studies by Murray and Paller [103] documented the existence of a vasoconstrictor defect in rats with cirrhosis and portal hypertension that they attributed to a post-receptor defect. Subsequent studies suggested that glucagon concentrations, similar to those measured in plasma of portal hypertensive subjects, attenuated both norepinephrine and vasopressin induced intestinal vasoconstriction [98,99,102]. Studies by Sitzmann et al. [90] reported similar findings for angiotensin II in the presence of prostacyclin. The

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aforementioned studies provided some of the first, albeit indirect evidence that cAMP-dependent vasodilators could interfere with vasoconstrictor function in portal hypertension. Since these initial observations, additional evidence supporting a role for cAMP as a mediator of portal hypertension-induced vascular dysfunction has become available. Huang et al. [104] reported elevations in cAMP and cGMP in arterial tissue from portal hypertensive rats. This finding established the fact that portal hypertension could lead to increases in vascular cell concentrations of cyclic nucleotides. A link between cAMP dependent events and reduced norepinephrine sensitivity has since been proposed by Wu and Benoit, [105]. In these studies, we assessed norepinephrine responsiveness of the intestinal microcirculation of normal and portal hypertensive rats before and after inhibition of protein kinase A by the competitive inhibitor Rp-cAMPs. Cyclic-GMP dependent pathways were investigated in similar experiments in which the soluble guanylyl cyclase was inhibited with LY-83583. PKA inhibition completely restored norepinephrine sensitivity in portal hypertensive rats. However, PKA inhibition had no effect on the intestinal vascular norepinephrine responsiveness of normal animals. Guanylyl cyclase inhibition, by LY-83583, did not correct the vasoconstrictor dysfunction associated with chronic portal hypertension. As a result of these studies, we believe that alterations in vascular contractile function in portal hypertension occur secondarily to elevation of cAMP and activation of protein kinase A. Additional evidence to support this contention has since been obtained using the synthetic somatostatin derivative, octreotide. In these studies, we showed that octreotide, at concentrations which are capable of inhibiting adenylyl cyclase activity, produced the same results as PKA inhibition [106]. These findings lead us to speculate that the therapeutic actions of octreotide in portal hypertensive patients is directly linked to its effects on cAMP synthesis. The cellular mechanism whereby cAMP dependent pathways impair vasoconstriction in chronic portal hypertension is still under investigation in our laboratory. In preliminary studies, we have simultaneously measured isometric contractile tension and cytosolic calcium concentration in norepinephrine-stimulated mesenteric arteries from normal and portal hypertensive rats. The ability of portal hypertensive vessels to respond to norepinephrine was significantly attenuated when compared with normal vessels. The reduced contractile tension in portal hypertensive vessels was associated with a lower cytosolic calcium concentration. To date, a direct link between cAMP and impaired calcium mobilization has not been proven in portal hypertensive resistance arteries. However, it is interesting to note that glucagon, at concentrations similar to those mea-

sured in the plasma of portal hypertensive animals, impairs calcium mobilization and tension development in normal mesenteric resistance arteries [107]. Future studies in the pathophysiology of portal hypertension should better define the role of cAMP in the associated vascular contractile dysfunction.

11. Closing remarks A major objective of this review has been to summarize a rapidly developing literature regarding the role of cyclic nucleotides in normal vascular smooth muscle function. In addition, we have provided evidence linking these second messengers to the vascular pathophysiology of portal hypertension. An emerging literature implicates alterations in cyclic nucleotide dependent events in a number of pathologic conditions including diabetes [108–112], pulmonary hypertension [113–115], arterial hypertension [116–118], impotence [109–111], inflammation [119–121], and ischemia/reperfusion injury [122–124]. However, the cellular mechanism defining the role of these agents in each of the aforementioned conditions is only partially understood. Continued investigation into the role of cyclic nucleotides as modulators of vascular cell function in normal and pathologic states will form the basis for refinement of pharmacological agents that target cardiovascular disease.

Acknowledgements This work is supported by a grant from the National Institutes of Health (DK-51430). A.M. McMahon and J.D. Gardner are recipients of Postdoctoral Fellowship Awards from the Southeast Consortium of the American Heart Association.

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