6.3. Mechanical stretch-induced ANP secretion

6.3.1. Role of thapsigargin-sensitive intracellular calcium pools

Changes in cytosolic free Ca²⁺ concentration constitute an important element of signal transduction in various cells (Clapham 1995). Several studies have addressed also the importance of intracellular Ca²⁺ concentration in stretch-induced ANP secretion. However, experiments by using various Ca²⁺ modulating agents such as nifedipine, caffeine and ryanodine have suggested both positive and negative modulation of stretch-stimulated ANP release by Ca²⁺ (Ruskoaho 1992).

SR is the major intracellular Ca²⁺ store in cardiac cells. It is sensitive to caffeine and ryanodine and has an important role in excitation-contraction coupling. Thapsigargin is a selective inhibitor of SERCA pumps and it is widely used as pharmacological tool for studying importance of these pumps (Thastrup 1990, Lytton & Nigam 1992). Recent studies have provided evidence for the presence of an IP₃- and thapsigargin-sensitive intracellular Ca²⁺ pool in atrial cells (Vigne et al. 1992, Negretti et al. 1993, Lewartowski et al. 1994). Vigne and co-workers (1992) showed that in atrial cells thapsigargin at the concentrations of 0.1-10 mol/L increased intracellular Ca²⁺ concentration in a manner that was independent of the presence of external Ca²⁺ and of the production of inositol phosphates. In agreement with the observation that thapsigargin rapidly raises intracellular Ca²⁺ concentration in cardiac cells (Vigne et al. 1992) and vascular cells (Thastrup 1990), the present results show that addition of thapsigargin to the perfusate at the concentrations above 100 nmol/L increases contractile force and ANP secretion and produces coronary vasoconstriction in the perfused rat heart preparation, suggesting that mobilization of Ca²⁺ from IP₃-sensititive intracellular stores may be involved in this response.

In order to examine the role of intracellular Ca²⁺ pools in mechanotransduction of cardiac myocytes low doses of thapsigargin (30 and 100 nmol/L) were infused to the perfusate for 25 min to deplete the thapsigargin-sensitive Ca²⁺ stores. Present results show that thapsigargin completely blocked stretch-activated ANP exocytosis from right atrial myocytes. This finding suggests that the stretch-induced increase in ANP secretion is linked to its capacity to mobilize a thapsigargin-sensitive intracellular Ca²⁺ pool. Present data, together with those from previous studies, also support the conclusion that there is a principal difference between the mechanical stretch- and agonist-induced ANP secretion, because thapsigargin has no influence on ANP secretion stimulated by ET-1 in cardiac cell cultures (Doubell & Thibault 1994).

6.3.2. Role of protein kinases

Mechanical stretch of cardiac myocytes in vitro causes an activation of multiple second messenger systems including PTKs and PKC (Sadoshima & Izumo 1993a). Peptide growth factors act by binding to and activating specific receptors with intrinsic PTK activity (Ullrich & Schlessinger 1990, Van der Geer & Hunter 1994). RPTKs have transmembrane segments, and some of the nonreceptor-type PTKs, such as Src family tyrosine kinases, are anchored to the inner surface of cell membranes. Thus it is possible that membrane stretch directly causes conformational change of tyrosine kinases, thereby activating them. Mechanical stretch caused activation of Src within 5 min in fetal lung cells (Liu et al. 1996) and an increase in tyrosine phoshorylation of FAK in mesangial cells (Hamasaki et al. 1995). In cardiac myocytes, mechanical stretch causes a significant increase in phosphotyrosine content of proteins, such as p42 and p44, within one minute (Sadoshima & Izumo 1993a). In cultured neonatal myocytes phorbol esters, ET-1 (Bogoyevitch et al. 1993) and mechanical stretch (Sadoshima & Izumo 1993a, 1997) have been shown to stimulate tyrosine phosphorylation of MAPKs, a family of related Ser/Thr kinases which activities are dependent on phosphorylation of both tyrosine and threonine residues (Anderson et al. 1990).

Several natural and synthetic PTK inhibitors have been used to study the physiological and pathophysiological role of these kinases. These inhibitors at micromolar concentrations have been shown to inhibit cell proliferation, DNA synthesis, proto-oncogene gene expression and phosphatidylinositol turnover caused by several growth factors (Lyall et al. 1989, Margolis et al. 1989, Hill et al. 1990, Levitzki 1992). Lavendustin A, a competitive inhibitor of ATP binding to the catalytic domain of PTKs (Onoda et al. 1989, 1990), was used to study the potential role of PTKs in regulation of mechanical stretch-induced ANP and BNP secretion. Present results show that lavendustin A dose-dependently decreased right atrial wall stretch-induced cardiac hormone secretion, while PKC inhibitor staurosporine, PKA, CaM kinase II and myosin light chain kinase inhibitors failed to block stretch-induced ANP secretion. Some PTK inhibitors may also inhibit other protein kinases (Levitzki 1992, Hidaka & Kobayashi 1992), e.g. PKC, which has a central role in the regulation of ANP secretion (Ruskoaho 1992). Previously, PKC inhibitors have been reported to either decrease (Page et al. 1991) or have no effect (Ishida et al. 1988) on stretch-induced ANP secretion in isolated rat atria preparations. In addition, PKC inhibitor staurosporine at a low concentration (10 nmol/L) was a potent inhibitor of ANP secretion produced by passive left ventricular wall stretch, suggesting that a PKC dependent pathway may play an important role in the regulation of ventricular stretch-stimulated ANP exocytosis (Kinnunen et al. 1993). However, it is unlikely that the effects of lavendustin A were non-specific because at the concentration used (maximally 1.3 mol/L) lavendustin A had no effects on basal cardiac hormone secretion or cardiac function, and this inhibition occurred at the concentrations similar to or even below those shown to inhibit the activities of PTKs in vitro (Onoda et al. 1989, 1990). Furthermore, lavendustin A failed to decrease TPA-induced ANP secretion, indicating that lavendustin A has no influence on PKC-mediated responses in this experimental model.

The mechanisms of tyrosine kinase activation by mechanical wall stress as well as the following activation of downstream signaling pathways are yet unclear. There may be hierarchy in protein kinase activation induced by mechanical forces and some protein kinases may have a regulatory role and some may have an obligatory role. In agreement with our results, hypotonic swelling-induced c-fos gene expression was abolished by PTK inhibitors but not by inhibitors of PKC and PLC (Sadoshima et al. 1996, Sadoshima & Izumo 1997) while stretch-induced induction was inhibited by inhibitors of PTKs, PKC and PLC. Thus, although hypotonic cell swelling and linear stretch activate separate signaling mechanisms, PTK activation is required for c-fos induction by both stimuli. PKC and PTK activities may both also be involved in coupling cardiac overload to alterations in atrial BNP synthesis, since lavendustin A and staurosporine inhibited stretch-induced increase in atrial BNP concentrations in perfused rat hearts (Magga et al. 1997b). Thus, although mechanical stretch activates multiple signaling mechanisms in the heart, specific protein kinase pathways seem to be important for different cellular processes, and of those pathways, PTK activity appears to be required for wall stretch-induced ANP and BNP exocytosis. It remains to be determined, however, which tyrosine kinases are responsible for wall stretch-induced cardiac hormone secretion.

6.3.3. Role of protein phosphatases

The finding that okadaic acid accelerated ANP secretion suggests that PPs may play a regulatory role in mechanical stretch-induced cardiac hormone exocytosis from atrial myocytes, possibly by dephosphorylating signaling molecules activated by PTKs. Several components of ERK1/ERK2 pathways are subject to regulation by PPA2, which causes dephosphorylation of threonine and inhibition of kinase activity (Bokemeyer et al. 1996). Previously okadaic acid has been shown to inhibit PPs in the heart at the same concentration range as was used in our studies (Neumann et al. 1993). Our finding that okadaic acid, a potent inhibitor of PPA2 and a strong inhibitor of PP1 (Cohen et al. 1990), can accelerate wall stretch-induced ANP secretion suggests that wall stretch-induced ANP secretion may involve activation of PTK pathway modulated by MAPK/ERK pathways, although many other possibilities also exist. Nevertheless, because the only targets

of okadaic acid are the catalytic subunits of PPA2 and PP1 (Cohen et al. 1990, Hunter 1995), these enzymes appear to play a significant role in atrial wall stretch-induced ANP secretion. Furthermore, the findings that okadaic acid accelerated and lavendustin A significantly decreased ANP secretion show that a balance between PTK and PP activities plays a major role in mechanical stretch-induced ANP exocytosis. On the basis of these studies one may suggest that tyrosine kinase or kinase cascade is required for the induction of cardiac hormone secretion by mechanical stretch, while the PKC and phospholipid system seems to be more important in agonist-induced hormone release. A hypothetical model of cellular mechanisms by which some protein kinases and phosphatases as well as thapsigargin-sensitive Ca²⁺ pool may regulate mechanical wall stretch- and segretagogue-induced ANP exocytosis is shown in Fig. 10.

6.3.4. Role of the endothelial factors

Previous studies have shown that mechanical stretch causes release of factor(s) to the culture medium, which in turn induces c-fos expression and activates MAPKs (Sadoshima & Izumo 1993a). Endogenous paracrine/autocrine factors such as Ang II and ET-1 liberated in response to mechanical stretch rather than direct stretch appear to be responsible for the activation of cardiac gene expression in neonatal ventricular myocytes (Yamasaki et al. 1995, Sadoshima & Izumo 1997). Several studies have demonstrated that ET-1 and Ang II signal through the PTK-dependent mechanism (Force et al. 1991, Simonson & Herman 1993). However, because the release of ANP by mechanical stretch takes place in the presence of treatment with an Ang II antagonist, losartan (Leskinen et al. 1997b), it is unlikely that Ang II is involved in mediating the wall stretch-induced ANP secretion observed in the present studies.

Recent studies suggest that cardiac contractile performance may be influenced by NO released by endothelial cells, analogous to vascular endothelial regulation of vessel tone and blood flow. Several actions of NO on myocardial contractile performance have been reported, including mediation of cytokine (Finkel et al. 1992) and cholinergic responses (Balligand et al. 1993), modulation of β -adrenergic inotropic responses (Balligand et al. 1993) and changes in myocardial relaxation, force-frequency relationship and Frank-Starling response (Smith et al. 1991, Brady et al. 1993, Prendengast et al. 1997). Our results showed that potent NOS inhibitor L-NAME (Rees et al. 1990) alone had no effect on basal contractile force or perfusion pressure, but it increased coronary perfusion pressure in the presence of ET-1, known to release NO in several models (Rubanyi & Polokoff 1994). These results agree with previous studies suggesting that vasoconstrictor influence is required for basal NO release (Adeagbo et al. 1994) and that cardiac myocytes do not alter myocardial contractility and synthesize appreciable amounts of constitutive NO in unstimulated conditions (Amrani et al. 1992, Balligand et al. 1993, Weyrich et al. 1994). Although there is significant evidence that NO may act on myocardial cells to regulate contractility, its role in the regulation of cardiac hormone exocytosis is less clear.

In support of the hypothesis that NO may be involved in the regulation of ANP release, bovine aortic cells, when placed in co-culture with rat atrial myocytes, stimulate the release of ANP and this release could be inhibited by acetylcholine (Lew & Baertschi 1989). Infusion of substances eliminating the action of NO (methylene blue, oxyhemoglobin or hydroquinone) also significantly increased the basal release of ANP from isolated rat atria (Sanchez-Ferrer et al. 1990). NO blocking agents have also been reported to overcome the inhibitory effect of acetylcholine on ANP secretion in vitro (Melo et al. 1996, Skvorak & Dietz 1997). We found that both L-NAME as well as L-arginine, a substrate for NOS, alone had no effects on basal ANP secretion in the perfused rat heart. This lack of effect may be due to the fact that under these experimental conditions coronary vessels are maximally dilated (Mäntymaa et al. 1990) and the basal production of NO is therefore minimal (Davies 1995). These results are consistent with those in the perfused atria, in which stimulation of NO production with acetylcholine or inhibition of NO had no significant effect on ANP secretion (Skvorak & Dietz 1997).

Leskinen and co-workers (1995) noted that in conscious rats the elevation of plasma ANP levels in response to acute volume expansion with 0.9 % saline was greater in the presence of L-NAME than in the control group, suggesting that NO may have a regulatory role in mechanical stretch-induced ANP secretion in vivo. In the perfused rat atria, the inhibition of NO activity has been shown to restore the normal response to stretch in the presence of acetylcholine (Skvorak & Dietz 1997). In the present study neither NOS inhibitor L-NAME or L-arginine alone had any effect on stretch-induced ANP secretion. In the presence of ET-1, L-NAME significantly increased right atrial wall stretch-induced ANP secretion. Since L-arginine partially reversed the effects of L-NAME on acute volume load-induced ANP release (Leskinen et al. 1995), NO released from the endothelium appears to inhibit tonically the secretion of ANP from cardiac myocytes in the presence of ET-1. Although cardiac cells themselves are capable of producing NO (Kelly et al. 1996, Winegrad 1997), this chemical signal may still originate from other cell types closely surrounding the myocytes in the atrium wall, including endothelial and endocardial cells.

In the perfused rat heart preparation ET-1 increases both basal and atrial wall stretch-induced ANP secretion (Mäntymaa et al. 1990, Shirakami et al. 1993). One possibility is that the atrial wall stretch could increase ET-1 release, which then acts on atrial myocytes to stimulate ANP release. Recently it has been shown that endothelial cells may to some extent contain stores of ET (MacClellan et al. 1993, Macarthur et al. 1994), which after its release can regulate the contractility of the heart (MacClellan et al. 1993). Local wall stretch caused by atrial stretch could release this stored ET, which may then participate in the regulation of ANP and BNP release. In cultured myocytes, the effect of ET-1 on ANP secretion has been shown to be mediated by endothelin ET_A-receptors (Thibault et al. 1994). Recently, the potent endothelin ET_A/B-receptor antagonist bosentan (Clozel et al. 1994) has been shown to significantly inhibit acute volume load-induced ANP secretion in conscious rats (Leskinen et al. 1997b). Present results show that bosentan had no effect on acute atrial wall stretch-induced ANP in the isolated perfused rat heart. These results are not consistent with recent in vivo studies (Leskinen et al. 1997b). The reason for these discrepant results is not clear, but one possibility is that after a one-hour stabilization period endothelial cells may not be able to produce, store and liberate ET-1 sufficiently, as suggested previously (Macarthur et al. 1994).

One possibility is that ET-1 regulates atrial stretch-induced cardiac hormone release via stimulation of ET_B-receptors, which leads to release of NO, and prostacyclin. To examine this question further, we used sarafotoxin 6C, a potent and selective endothelin ET_B-receptor agonist (Bax & Saxena 1994), to stimulate NOS activity during atrial stretch. Sarafotoxin 6C had no effects on basal or stretch-induced ANP secretion, supporting the important role of endothelin ET_A-receptors in ANP secretion. Taken together, these results show that the atrial wall stretch-induced ANP release under these experimental conditions appears to occur independently of ET-1 and implicate that the mechanism for NO formation is local wall stretch rather than the release of ET-1 and consequent stimulation of endothelin ET_B-receptors. These experiments, however, do not exclude the possibility that other autocrine and/or paracrine factors are released which may be capable of stimulating wall stretch-induced cardiac hormone secretion. A hypothetical model of mechanisms by which ET-1 and NO may regulate ANP release is shown in Fig. 11.