[Frontiers in Bioscience 3, d194-207, February 15, 1998]

	[Frontiers in Bioscience 3, d194-207, February 15, 1998] Reprints PubMed CAVEAT LECTOR
	VASOPRESSIN SIGNALING PATHWAYS IN VASCULAR SMOOTH MUSCLE Raphael A. Nemenoff Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262 Received 1/21/98 Accepted 2/4/98 4. POST-RECEPTOR SIGNALING PATHWAYS 4.1. Mobilization of intracellular Ca²⁺ Exposure of VSMC to AVP rapidly results in increases in intracellular Ca²⁺([Ca²⁺]i). This increase is mediated through the V1 receptor, since several studies have shown that specific inhibitors of the V1 receptor antagonize the effect (6, 11). The rise in [Ca²⁺]i is not inhibited by pretreatment of cells with pertussis toxin, indicating that the V1 receptor, via a pertussis-insensitive G-protein, probably Gq, causes activation of phosphatidylinositol-specific phospholipase C-beta (PLC-beta) (30), resulting in production of inositol trisphosphate, which releases Ca²⁺ from intracellular stores, (31, 32, 33) and diacylglycerol which activates protein kinase C (PKC). While this rapid increase in [Ca²⁺]i is due to release of intracellular stores, a second phase of Ca²⁺ increase involves receptor-mediated Ca²⁺ influx through cation channels. This phase is slower and more sustained and appears to involve activation of Ca²⁺-permeable nonselective cation channels (34). Consistent with this finding, several groups have shown that AVP stimulates Ca²⁺ entry as assessed by 45 Ca²⁺ uptake (35, 36). One area of future research will be to determine the post-receptor signaling events leading to Ca²⁺ entry in these cells. Ca²⁺-mediated Ca²⁺ entry is likely to play a role, but other effectors also contribute. The rapid increase in [Ca²⁺]i is the critical event in the initial contractile response to AVP. Elevated Ca²⁺ levels, through the action of calmodulin, activate myosin light chain kinase (MLCK), which in turn phosphorylates myosin light chain (37), leading to enhanced actinomyosin ATPase activity and contraction. In fact, myosin phosphorylation has been employed as a quantitative marker for contraction (38). While the rapid increase in [Ca²⁺]i is transient, contractile force is maintained for longer periods of time. The molecular events controlling this so-called "latch" state (37) are still poorly understood. An additional control of contractile force involves alterations in the force/Ca²⁺ ratio, or Ca²⁺ sensitization. A role for low molecular weight G-proteins of the Rho family has been proposed to play a role in this process. Activation of Rho leads to increased Ca²⁺ sensitization through novel downstream kinases which phosphorylate and inactivate myosin light chain phosphatase (39). The ability of vasoconstrictors such as AVP to modulate this pathway remains to be examined, and it is likely that AVP will regulate contraction at multiple points. Several other Ca²⁺-dependent enzymes will also be activated in response to elevations in [Ca²⁺]i. Ca²⁺ dependent forms of adenylyl cyclase have been described in VSMC (40). In the setting of AVP-mediated rises in [Ca²⁺]i, cAMP levels will be increased, leading to activation of protein kinase A, which phosphorylates MLCK and decreases the affinity of this enzyme for calmodulin(38) This will result in a decreased contraction at a given Ca²⁺ concentration, antagonizing the contractile effect of AVP. In addition, cAMP which has antimitogenic activity and decreases expression of muscle-specific genes in VSMC (41), will act as a negative feedback to counteract the hypertrophic response to AVP. Increased Ca²⁺ will also, via calmodulin, activate other Ca²⁺-dependent kinases such as Ca²⁺/CaM kinase II (42). The physiologic role of this kinase in VSMC is not well understood. However, at least one study has shown that CaM kinase II is involved in the activation of the extracellular-regulated kinases (ERKs), which are members of the mitogen-activated protein kinase family (see below). Other targets of this pathway may include transcription factors which remain to be identified. Lastly, elevations in [Ca²⁺]i are critical for the activation of phospholipase A2 (43), which is the rate limiting enzyme in the production of eicosanoids. We will consider this pathway in greater detail below. In summary, AVP rapidly engages multiple pathways leading to both acute and sustained increases in [Ca²⁺]i which are critical events in the vasoconstrictor effects of this hormone. Moreover, activation of multiple Ca²⁺-dependent pathways will in all likelihood also impinge on the longer term effects of AVP on growth and differentiation. 4.2. Regulation of protein kinase pathways Protein phosphorylation is perhaps the most common post-translational modification in eukaryotic cells, and is regulated through multiple families of protein kinases and phosphatases. In VSMC, AVP rapidly activates multiple protein kinase cascades. Much work has recently focused on the mitogen activated protein (MAP) kinase family of kinases. This family of proline-directed kinases are highly conserved from yeast to man (44). Three major branches of the MAP kinase family have been identified. These include the extracellular regulated kinases (ERKs), the first members identified, which were previously designated p42/p44 MAP kinase; the stress-activated protein kinases/c-Jun amino terminal kinases (SAPK/JNK), and the p38 MAP kinase family which have homology to the HOG enzymes in yeast. A large number of review articles have recently appeared describing the regulation of these kinases (45, 46, 47, 48, 49, 50, 51, 52) , so this article will summarize these findings and focus on these pathways in VSMC. ERK activation by receptor tyrosine kinases is mediated through the low molecular weight G protein Ras. Phosphorylation of receptors such as the PDGF receptor recruits specific effector molecules through binding of SH2 domains to specific phosphotyrosine residues on the cytoplasmic tail of the receptor. The binding to SOS, via the linker GRB-2 promotes displacement of GDP by GTP on the low molecular weight G-protein Ras, resulting in activation (53). Activation of Ras then initiates a cascade of protein kinases including the protooncogene serine kinase Raf-1, and the dual specificity kinase MEK-1, culminating in the activation of ERK family members p42/44 MAP kinase (49, 54, 55, 56, 57, 58). We have demonstrated that this pathway is operative in VSMC stimulated by PDGF or EGF (28). Vasoconstrictors also activate ERK. In VSMC, AVP stimulates ERK activity to the same extent as PDGF (5-10 fold), and with similar kinetics. ERK activation is detectable within 2 minutes after stimulation, is maximal by 5-10 minutes, and has returned to basal levels by 30 minutes (27, 59). However, direct measurement of Ras activation assessed by the ratio of GTP/GDP bound, found that AVP did not significantly activate Ras compared to EGF or PDGF(28). Similarly, activation of Raf as determined by phosphorylation of MEK-1, was much weaker with AVP than with receptor tyrosine kinases. Activation of ERKs in VSMC by AVP did require activation of protein kinase C (59, 60). Inhibition of PKC by either pharmacological agents, or by down regulation following chronic exposure to high concentrations of phorbol esters, completely abolished the ability of AVP to activate ERKs, while not affecting activation by PDGF or EGF. Thus while activation of ERKs in response to AVP is similar to that seen with PDGF, the signaling pathways are different. In other cell types ERK activation is critical for cell growth (61). Following cell stimulation, ERKs translocate to the nucleus where they presumably phosphorylate transcription factors. It has been shown that Elk-1, a member of the ets family of transcription factors is a substrate of ERKs (52, 62). ERKs also phsophorylate cytoplasmic enzymes including cytosolic PLA2 (63). Based on analogies with other cell types, it appears reasonable to assume that ERK activation by AVP contributes to the hypertrophic growth response of these cells. Use of recently developed specific MEK-1 inhibitors (64) will be able to clarify this role. The SAPK/JNK family of kinases was originally identified as kinases activated by external stresses such as UV light or hyperosmolarity (65, 66, 67, 68, 69, 70). By molecular cloning three genes encoding distinct JNK family members have been characterized (69, 71, 72, 73). Due to the expression of splice variants, at least 10 distinct JNKs have been identified (74). As their name indicates, these kinases phosphorylate the amino terminal of the transcription factor c-Jun, which results in activation of transcription at AP-1 sites. In addition, JNKs can phosphorylate other transcription factors such as ATF-2 and Elk-1(75). Analogously to the ERKs, JNKs are activated by upstream dual specificity kinases which phosphorylate threonine and tyrosine residues in the sequence TPF, conserved in all the JNKs (66, 76). AVP, as well as angiotensin II rapidly activate JNKs in VSMC (29). Activation of JNKs has a slower time course than that for the ERK pathway, with maximal activation (4-6-fold) detected at approximately 15 minutes, and values returning to basal levels by 60 minutes. The activation appears to be mediated by members of the Gq family of G-proteins, since stable expression of constitutive forms of Gq family alpha-subunits resulted in constitutive activation of JNKs (29). In contrast to ERK activation, stimulation of JNK activity does not appear to be mediated through a PKC dependent pathway. Phorbol esters, which potently stimulate ERKs are weak activators of JNK in VSMC. Inhibition of PKC using pharmacological agents or down regulation by chronic exposure to high concentrations of phorbol esters did not significantly affect JNK activation. However, inhibiting agonist-mediated increases in [Ca²⁺]i by pretreatment of cells with the Ca²⁺chelator BAPTA blocked AVP-stimulated JNK activity. Exposure to ionomycin did not by itself activate JNKs, indicating that Ca²⁺ is necessary but not sufficient for JNK activation. The physiologic role of JNK remain to be determined. We will describe recent studies from our laboratory below suggesting a role for this pathway in mediating increased transcription of SM-alpha-actin. A third branch of the MAP kinase family is represented by p38 MAP kinase. The yeast homologue of this enzyme is involved in response to hyperosmotic shock (77). The effects of vasoconstrictors and growth factors on p38 activation in VSMC has not been extensively examined. However, G-protein coupled receptors activate p38 MAP kinase in other cell types (78). The physiologic targets of p38 are not well characterized. The availability of specific inhibitors of this pathway (79) will facilitate understanding the role of these kinases in VSMC. 4.3. Regulation of arachidonic acid release and eicosanoid production Arachidonic acid and its metabolites have been shown to have a variety of roles in different cell types. In VSMC, stimulation by AVP or other vasoconstrictors results in the production of vasodilatory prostaglandins, principally prostaglandin E1(PGE1) and prostacyclin (PGI2) (80). These eicosanoids are released from the cell and act in an autocrine fashion through specific G-protein coupled receptors. In VSMC, these prostaglandins through Gs activate adenylyl cyclase and stimulate production of cAMP (81). Elevated cAMP levels, as discussed above, inhibit contraction and proliferation of VSMC. Thus these eicosanoids provide a negative feedback mechanism to counteract the effects of vasoconstrictors such as AVP. The rate limiting step in eicosanoid production is the release of free arachidonic acid from membrane phospholipids. The bulk of arachidonic acid in a resting cell is esterified to the sn-2 position of membrane phospholipids. While multiple pathways can mediate hydrolysis of the ester bond to produce free arachidonic acid, the dominant pathway in most cells including VSMC is through the action of phospholipase A2(PLA2). Multiple forms of this enzyme exist which can be dissociated into secreted forms and intracellular forms. Several years ago we and others identified a major form of PLA2 which controls agonist-induced arachidonic acid in many cell types(82, 83, 84, 85.) This enzyme, now designated cPLA2, is an 85 kDa enzyme which is specific for arachidonic acid. cPLA2 requires Ca²⁺ for enzymatic activity and translocates to an intracellular compartment in response to elevation in [Ca²⁺]i (43, 86). By immunofluorescence and electron microscopy it has been show that cPLA2 localizes to the nuclear envelope(87). In addition to Ca²⁺, cPLA2 is regulated by protein phosphorylation, and is phosphorylated and activated by ERKs (63, 88). In VSMC, cPLA2 is constitutively expressed, and acutely activated by AVP as well as other growth factors such as PDGF. However, recent data indicate that other forms of PLA2 may play a role in eicosanoid production in VSMC. Gross and coworkers have described a Ca²⁺-independent form of PLA2(89). Employing a specific inhibitor of this enzyme, these workers have reported that approximately two thirds of AVP-induced arachidonic acid release in VSMC is contributed by this enzyme (90). The signals mediating AVP-regulation of this enzyme remain to be fully determined. However, depletion of intracellular Ca²⁺ stores may play a role (91). The relative contributions of these distinct isoforms of PLA2 to prostaglandin production and vascular smooth muscle physiology is also an area for future research. Prostaglandin production is mediated through the cyclooxygenase (COX) family of enzymes. COX-1 is generally localized to the endoplasmic reticulum and is constitutively expressed in most cell types including VSMC. COX-2, which was originally identified as an immediate early response gene (92), is induced by mitogenic stimuli. The rapid production of prostaglandins in response to AVP is likely mediated through COX-1, since COX-2 levels are very low in resting VSMC. In fibroblasts, PDGF stimulates induction of COX-2 expression through increased transcription (93). We have observed that PDGF also induces COX-2 expression and constitutive increases in prostaglandin production in VSMC (28); this induction is not observed with AVP treatment, and may play a role in the suppression of muscle-specific gene expression mediated by PDGF. It is therefore apparent that multiple enzyme pathways mediate prostaglandin production in VSMC, and that these are differentially regulated by AVP vis a vis PDGF. Like many cells, VSMC produce a spectrum of eicosanoids, of which PGE1 and PGI-2 are the major secreted products. Eicosanoids produced in lesser quantities may have important physiologic roles in these cells. To that end, it has been reported that prostaglandins A and J are potent antimitogenic agents in these cells (94). Production of these molecules in response to AVP or other agonists has not been examined. An additional complexity in this field is the recent finding that activation of cPLA2 appears to be critical for growth of VSMC (95). Exposure of cells to antisense oligonucleotides inhibited growth. To date, no eicosanoids have been detected in VSMC which promote growth of these cells, therefore the critical role for PLA2 in proliferation is not well understood. It is conceivable that activation of PLA2 has effects distinct from eicosanoid production. One report has indicated that arachidonic acid activates mitogen activated protein kinase pathways in VSMC (96). Thus PLA2 may lie both upstream and downstream of the MAP kinase pathway. Besides activating phospholipases A2 and C, AVP also stimulates phospholipase D (PLD) activity in VSMC (97). This enzyme hydrolyzes membrane phospholipids to produce phosphatidic acid, which in turn can be converted to diglyceride through the sequential action of phosphatidic acid phosphohydrolases (98, 99). Diglyceride production in VSMC and other cells is composed of an acute and a sustained phase. PLD is likely to be responsible for the sustained phase of diglyceride production, and may thus contribute to long-term activation of PKC. A number of physiologic roles have been attributed to phosphatidic acid. Both phosphatidic acid and lysophosphatidic acid have mitogenic effects in fibroblasts (100, 101) . The role that PLD products play in VSMC physiology has not been established. Similarly very little is known about the PLD isoforms or its mechanisms of regulation in VSMC.

[Frontiers in Bioscience 3, d194-207, February 15, 1998]
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CAVEAT LECTOR

VASOPRESSIN SIGNALING PATHWAYS IN VASCULAR SMOOTH MUSCLE

Raphael A. Nemenoff

Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262

Received 1/21/98 Accepted 2/4/98

4. POST-RECEPTOR SIGNALING PATHWAYS

4.1. Mobilization of intracellular Ca²⁺

Exposure of VSMC to AVP rapidly results in increases in intracellular Ca²⁺([Ca²⁺]i). This increase is mediated through the V1 receptor, since several studies have shown that specific inhibitors of the V1 receptor antagonize the effect (6, 11). The rise in [Ca²⁺]i is not inhibited by pretreatment of cells with pertussis toxin, indicating that the V1 receptor, via a pertussis-insensitive G-protein, probably Gq, causes activation of phosphatidylinositol-specific phospholipase C-beta (PLC-beta) (30), resulting in production of inositol trisphosphate, which releases Ca²⁺ from intracellular stores, (31, 32, 33) and diacylglycerol which activates protein kinase C (PKC). While this rapid increase in [Ca²⁺]i is due to release of intracellular stores, a second phase of Ca²⁺ increase involves receptor-mediated Ca²⁺ influx through cation channels. This phase is slower and more sustained and appears to involve activation of Ca²⁺-permeable nonselective cation channels (34). Consistent with this finding, several groups have shown that AVP stimulates Ca²⁺ entry as assessed by 45 Ca²⁺ uptake (35, 36). One area of future research will be to determine the post-receptor signaling events leading to Ca²⁺ entry in these cells. Ca²⁺-mediated Ca²⁺ entry is likely to play a role, but other effectors also contribute.

The rapid increase in [Ca²⁺]i is the critical event in the initial contractile response to AVP. Elevated Ca²⁺ levels, through the action of calmodulin, activate myosin light chain kinase (MLCK), which in turn phosphorylates myosin light chain (37), leading to enhanced actinomyosin ATPase activity and contraction. In fact, myosin phosphorylation has been employed as a quantitative marker for contraction (38). While the rapid increase in [Ca²⁺]i is transient, contractile force is maintained for longer periods of time. The molecular events controlling this so-called "latch" state (37) are still poorly understood. An additional control of contractile force involves alterations in the force/Ca²⁺ ratio, or Ca²⁺ sensitization. A role for low molecular weight G-proteins of the Rho family has been proposed to play a role in this process. Activation of Rho leads to increased Ca²⁺ sensitization through novel downstream kinases which phosphorylate and inactivate myosin light chain phosphatase (39). The ability of vasoconstrictors such as AVP to modulate this pathway remains to be examined, and it is likely that AVP will regulate contraction at multiple points.

Several other Ca²⁺-dependent enzymes will also be activated in response to elevations in [Ca²⁺]i. Ca²⁺ dependent forms of adenylyl cyclase have been described in VSMC (40). In the setting of AVP-mediated rises in [Ca²⁺]i, cAMP levels will be increased, leading to activation of protein kinase A, which phosphorylates MLCK and decreases the affinity of this enzyme for calmodulin(38) This will result in a decreased contraction at a given Ca²⁺ concentration, antagonizing the contractile effect of AVP. In addition, cAMP which has antimitogenic activity and decreases expression of muscle-specific genes in VSMC (41), will act as a negative feedback to counteract the hypertrophic response to AVP.

Increased Ca²⁺ will also, via calmodulin, activate other Ca²⁺-dependent kinases such as Ca²⁺/CaM kinase II (42). The physiologic role of this kinase in VSMC is not well understood. However, at least one study has shown that CaM kinase II is involved in the activation of the extracellular-regulated kinases (ERKs), which are members of the mitogen-activated protein kinase family (see below). Other targets of this pathway may include transcription factors which remain to be identified. Lastly, elevations in [Ca²⁺]i are critical for the activation of phospholipase A2 (43), which is the rate limiting enzyme in the production of eicosanoids. We will consider this pathway in greater detail below. In summary, AVP rapidly engages multiple pathways leading to both acute and sustained increases in [Ca²⁺]i which are critical events in the vasoconstrictor effects of this hormone. Moreover, activation of multiple Ca²⁺-dependent pathways will in all likelihood also impinge on the longer term effects of AVP on growth and differentiation.

4.2. Regulation of protein kinase pathways

Protein phosphorylation is perhaps the most common post-translational modification in eukaryotic cells, and is regulated through multiple families of protein kinases and phosphatases. In VSMC, AVP rapidly activates multiple protein kinase cascades. Much work has recently focused on the mitogen activated protein (MAP) kinase family of kinases. This family of proline-directed kinases are highly conserved from yeast to man (44). Three major branches of the MAP kinase family have been identified. These include the extracellular regulated kinases (ERKs), the first members identified, which were previously designated p42/p44 MAP kinase; the stress-activated protein kinases/c-Jun amino terminal kinases (SAPK/JNK), and the p38 MAP kinase family which have homology to the HOG enzymes in yeast. A large number of review articles have recently appeared describing the regulation of these kinases (45, 46, 47, 48, 49, 50, 51, 52) , so this article will summarize these findings and focus on these pathways in VSMC.

ERK activation by receptor tyrosine kinases is mediated through the low molecular weight G protein Ras. Phosphorylation of receptors such as the PDGF receptor recruits specific effector molecules through binding of SH2 domains to specific phosphotyrosine residues on the cytoplasmic tail of the receptor. The binding to SOS, via the linker GRB-2 promotes displacement of GDP by GTP on the low molecular weight G-protein Ras, resulting in activation (53). Activation of Ras then initiates a cascade of protein kinases including the protooncogene serine kinase Raf-1, and the dual specificity kinase MEK-1, culminating in the activation of ERK family members p42/44 MAP kinase (49, 54, 55, 56, 57, 58). We have demonstrated that this pathway is operative in VSMC stimulated by PDGF or EGF (28).

Vasoconstrictors also activate ERK. In VSMC, AVP stimulates ERK activity to the same extent as PDGF (5-10 fold), and with similar kinetics. ERK activation is detectable within 2 minutes after stimulation, is maximal by 5-10 minutes, and has returned to basal levels by 30 minutes (27, 59). However, direct measurement of Ras activation assessed by the ratio of GTP/GDP bound, found that AVP did not significantly activate Ras compared to EGF or PDGF(28). Similarly, activation of Raf as determined by phosphorylation of MEK-1, was much weaker with AVP than with receptor tyrosine kinases. Activation of ERKs in VSMC by AVP did require activation of protein kinase C (59, 60). Inhibition of PKC by either pharmacological agents, or by down regulation following chronic exposure to high concentrations of phorbol esters, completely abolished the ability of AVP to activate ERKs, while not affecting activation by PDGF or EGF. Thus while activation of ERKs in response to AVP is similar to that seen with PDGF, the signaling pathways are different.

In other cell types ERK activation is critical for cell growth (61). Following cell stimulation, ERKs translocate to the nucleus where they presumably phosphorylate transcription factors. It has been shown that Elk-1, a member of the ets family of transcription factors is a substrate of ERKs (52, 62). ERKs also phsophorylate cytoplasmic enzymes including cytosolic PLA2 (63). Based on analogies with other cell types, it appears reasonable to assume that ERK activation by AVP contributes to the hypertrophic growth response of these cells. Use of recently developed specific MEK-1 inhibitors (64) will be able to clarify this role.

The SAPK/JNK family of kinases was originally identified as kinases activated by external stresses such as UV light or hyperosmolarity (65, 66, 67, 68, 69, 70). By molecular cloning three genes encoding distinct JNK family members have been characterized (69, 71, 72, 73). Due to the expression of splice variants, at least 10 distinct JNKs have been identified (74). As their name indicates, these kinases phosphorylate the amino terminal of the transcription factor c-Jun, which results in activation of transcription at AP-1 sites. In addition, JNKs can phosphorylate other transcription factors such as ATF-2 and Elk-1(75). Analogously to the ERKs, JNKs are activated by upstream dual specificity kinases which phosphorylate threonine and tyrosine residues in the sequence TPF, conserved in all the JNKs (66, 76). AVP, as well as angiotensin II rapidly activate JNKs in VSMC (29). Activation of JNKs has a slower time course than that for the ERK pathway, with maximal activation (4-6-fold) detected at approximately 15 minutes, and values returning to basal levels by 60 minutes. The activation appears to be mediated by members of the Gq family of G-proteins, since stable expression of constitutive forms of Gq family alpha-subunits resulted in constitutive activation of JNKs (29). In contrast to ERK activation, stimulation of JNK activity does not appear to be mediated through a PKC dependent pathway. Phorbol esters, which potently stimulate ERKs are weak activators of JNK in VSMC. Inhibition of PKC using pharmacological agents or down regulation by chronic exposure to high concentrations of phorbol esters did not significantly affect JNK activation. However, inhibiting agonist-mediated increases in [Ca²⁺]i by pretreatment of cells with the Ca²⁺chelator BAPTA blocked AVP-stimulated JNK activity. Exposure to ionomycin did not by itself activate JNKs, indicating that Ca²⁺ is necessary but not sufficient for JNK activation. The physiologic role of JNK remain to be determined. We will describe recent studies from our laboratory below suggesting a role for this pathway in mediating increased transcription of SM-alpha-actin.

A third branch of the MAP kinase family is represented by p38 MAP kinase. The yeast homologue of this enzyme is involved in response to hyperosmotic shock (77). The effects of vasoconstrictors and growth factors on p38 activation in VSMC has not been extensively examined. However, G-protein coupled receptors activate p38 MAP kinase in other cell types (78). The physiologic targets of p38 are not well characterized. The availability of specific inhibitors of this pathway (79) will facilitate understanding the role of these kinases in VSMC.

4.3. Regulation of arachidonic acid release and eicosanoid production

Arachidonic acid and its metabolites have been shown to have a variety of roles in different cell types. In VSMC, stimulation by AVP or other vasoconstrictors results in the production of vasodilatory prostaglandins, principally prostaglandin E1(PGE1) and prostacyclin (PGI2) (80). These eicosanoids are released from the cell and act in an autocrine fashion through specific G-protein coupled receptors. In VSMC, these prostaglandins through Gs activate adenylyl cyclase and stimulate production of cAMP (81). Elevated cAMP levels, as discussed above, inhibit contraction and proliferation of VSMC. Thus these eicosanoids provide a negative feedback mechanism to counteract the effects of vasoconstrictors such as AVP.

The rate limiting step in eicosanoid production is the release of free arachidonic acid from membrane phospholipids. The bulk of arachidonic acid in a resting cell is esterified to the sn-2 position of membrane phospholipids. While multiple pathways can mediate hydrolysis of the ester bond to produce free arachidonic acid, the dominant pathway in most cells including VSMC is through the action of phospholipase A2(PLA2). Multiple forms of this enzyme exist which can be dissociated into secreted forms and intracellular forms. Several years ago we and others identified a major form of PLA2 which controls agonist-induced arachidonic acid in many cell types(82, 83, 84, 85.) This enzyme, now designated cPLA2, is an 85 kDa enzyme which is specific for arachidonic acid. cPLA2 requires Ca²⁺ for enzymatic activity and translocates to an intracellular compartment in response to elevation in [Ca²⁺]i (43, 86). By immunofluorescence and electron microscopy it has been show that cPLA2 localizes to the nuclear envelope(87). In addition to Ca²⁺, cPLA2 is regulated by protein phosphorylation, and is phosphorylated and activated by ERKs (63, 88). In VSMC, cPLA2 is constitutively expressed, and acutely activated by AVP as well as other growth factors such as PDGF. However, recent data indicate that other forms of PLA2 may play a role in eicosanoid production in VSMC. Gross and coworkers have described a Ca²⁺-independent form of PLA2(89). Employing a specific inhibitor of this enzyme, these workers have reported that approximately two thirds of AVP-induced arachidonic acid release in VSMC is contributed by this enzyme (90). The signals mediating AVP-regulation of this enzyme remain to be fully determined. However, depletion of intracellular Ca²⁺ stores may play a role (91). The relative contributions of these distinct isoforms of PLA2 to prostaglandin production and vascular smooth muscle physiology is also an area for future research.

Prostaglandin production is mediated through the cyclooxygenase (COX) family of enzymes. COX-1 is generally localized to the endoplasmic reticulum and is constitutively expressed in most cell types including VSMC. COX-2, which was originally identified as an immediate early response gene (92), is induced by mitogenic stimuli. The rapid production of prostaglandins in response to AVP is likely mediated through COX-1, since COX-2 levels are very low in resting VSMC. In fibroblasts, PDGF stimulates induction of COX-2 expression through increased transcription (93). We have observed that PDGF also induces COX-2 expression and constitutive increases in prostaglandin production in VSMC (28); this induction is not observed with AVP treatment, and may play a role in the suppression of muscle-specific gene expression mediated by PDGF. It is therefore apparent that multiple enzyme pathways mediate prostaglandin production in VSMC, and that these are differentially regulated by AVP vis a vis PDGF.

Like many cells, VSMC produce a spectrum of eicosanoids, of which PGE1 and PGI-2 are the major secreted products. Eicosanoids produced in lesser quantities may have important physiologic roles in these cells. To that end, it has been reported that prostaglandins A and J are potent antimitogenic agents in these cells (94). Production of these molecules in response to AVP or other agonists has not been examined. An additional complexity in this field is the recent finding that activation of cPLA2 appears to be critical for growth of VSMC (95). Exposure of cells to antisense oligonucleotides inhibited growth. To date, no eicosanoids have been detected in VSMC which promote growth of these cells, therefore the critical role for PLA2 in proliferation is not well understood. It is conceivable that activation of PLA2 has effects distinct from eicosanoid production. One report has indicated that arachidonic acid activates mitogen activated protein kinase pathways in VSMC (96). Thus PLA2 may lie both upstream and downstream of the MAP kinase pathway.

Besides activating phospholipases A2 and C, AVP also stimulates phospholipase D (PLD) activity in VSMC (97). This enzyme hydrolyzes membrane phospholipids to produce phosphatidic acid, which in turn can be converted to diglyceride through the sequential action of phosphatidic acid phosphohydrolases (98, 99). Diglyceride production in VSMC and other cells is composed of an acute and a sustained phase. PLD is likely to be responsible for the sustained phase of diglyceride production, and may thus contribute to long-term activation of PKC. A number of physiologic roles have been attributed to phosphatidic acid. Both phosphatidic acid and lysophosphatidic acid have mitogenic effects in fibroblasts (100, 101) . The role that PLD products play in VSMC physiology has not been established. Similarly very little is known about the PLD isoforms or its mechanisms of regulation in VSMC.