Marcus Neusser

Transforming growth factor β1 modulates angiotensin induced calcium influx in vascular smooth muscle

Abstract. The modulatory effects of transforming growth factor β1 (TGF β1) on the angiotensin II (Ang II)‐induced increase in cytosolic free calcium concentration ([Ca2+]i) were investigated in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto rats (WKY). [Ca2+]i in VSMC was measured using the fluorescent dye fura‐2. When TGF β1 was applied 30 s prior to Ang II, the Ang II‐induced [Ca2+]i increase was significantly enhanced in VSMC from SHR (P < 0.05 compared to control), whereas after the preincubation with TGF β1 for 30 min, the Ang II‐induced [Ca2+]i increase was significantly reduced in VSMC from both strains. Using the manganese‐quenching technique, it was confirmed that short‐term exposure to TGF β1 enhanced the Ang II‐induced trans‐plasma‐membrane calcium influx in SHR. The inhibition of protein kinase C by calphostin C abolished the stimulatory effect of TGF β1 on the Ang II‐induced [Ca2+]i increase. It is concluded that TGF β1 modulates the Ang II‐induced calcium handling in VSMC.

Different calcium storage pools in vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats.

Objective To evaluate whether the distribution of intracellular free calcium may be impaired in primary hypertension. Design Cytosolic free calcium and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR). Methods The concentrations of intracellular and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats aged 6 months from the Münster strain (SHR) and from age-matched normotensive Wistar-Kyoto (WKY) rats. Vascular smooth muscle cells were grown on coverslips, and fluorescence measurements of the intracellular calcium concentration were performed using fura-2. The different effects of thapsigargin, a selective Ca-ATPase inhibitor, and of angiotensin II (Ang II) on the calcium storage pools were investigated. Results In the absence of external calcium thapsigargin produced a dose-dependent transient increase in the concentration of intracellular calcium in vascular smooth muscle cells. The thapsigargin-induced maximum peak increase in the concentration of intracellular calcium was not significantly different in SHR and WKY rats. After depletion of the thapsigargin-sensitive calcium pools the addition of 100nmol/l Ang II produced a rise in the concentration of intracellular calcium in vascular smooth muscle cells from SHR and WKY rats. Using vascular smooth muscle cells from the SHR the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the presence and absence of thapsigargin, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in vascular smooth muscle cells from SHR. In contrast, in the WKY rats the response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive pool. When Ang II and thapsigargin were administered in the reverse order, i.e. Ang II before thapsigargin, the thapsigargin response was diminished in the WKY rats but not in the SHR. Conclusion SHR differ from WKY rats in having vascular smooth muscle cells that contain thapsigargin-sensitive calcium storage pools that are distinct from the Ang II-sensitive calcium pools.

Effect of captopril on vasoconstriction and Ca2+ fluxes in aortic smooth muscle.

The effects of captopril on the response of cytosolic free Ca2+ concentration in cultured vascular smooth muscle cells of aortas from Wistar-Kyoto and spontaneously hypertensive rats to angiotensin II (Ang II) and bradykinin were studied using fura 2. Incubation with captopril for longer than 10 minutes caused a decreased response of cytosolic free Ca2+ to Ang II and bradykinin. Maximal effects of captopril were observed after a 40-minute incubation. The inhibitory effect of captopril was abolished in Ca(2+)-free medium, suggesting that captopril acts by blocking Ca2+ influx. Similar effects were observed with enalaprilat. Isometric contraction of aortic strips induced by Ang II in normotensive rats was reduced from 6.5 +/- 2.5 to 1.8 +/- 0.6 mN by a 40-minute incubation with 1 mumol/L captopril (P = .016). Enalaprilat similarly decreased the Ang II-induced contraction. Besides the inhibition of the angiotensin converting enzyme, direct effects of Ang II converting enzyme inhibitors on vascular contraction and Ca2+ influx in vascular smooth muscle cells may be of therapeutic relevance.

Effect of Inhibition of Sarcoplasmic Ca2+-ATPase on Vasoconstriotion and Cytosolic Ca2+ in Aortic Smooth Muscle from Spontaneously Hypertensive and Normotensive Rats

To evaluate the influence of the sarcoplasmic Ca(2+)-ATPase, isometric vasoconstrictions of aortic strips from spontaneously hypertensive rats from the Münster strain (SHR) and normotensive Wistar-Kyoto rats (WKY) were measured after inhibition of Ca(2+)-ATPase by thapsigargin. Inhibition of Ca(2+)-ATPase by thapsigargin caused a biphasic contractile response of the aorta in both SHR and WKY (maximum increase of tension: 1.7 +/- 0.3 x 10(-3) Newton and 2.1 +/- 0.3 x 10(-3) Newton, respectively; mean +/- SE). The second peak of the contractile response was abolished in the absence of external calcium or by inhibition of transplasmamembrane calcium influx by nifedipine, indicating that the second peak occurs as a consequence of calcium influx from the extracellular space. The initial peak of the contractile response after thapsigargin administration was abolished in the presence of an intracellular calcium antagonist, 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the initial response was due to calcium release from intracellular stores. Measurements using the fluorescent dye fura2 showed that thapsigargin increased the cytosolic free calcium concentration ([Ca2+]i) in SHR by 72.6 +/- 7.3 nmol/l (n = 34) and in WKY by 53.3 +/- 6.6 nmol/l (n = 39), showing no significant differences between the two strains. The inhibition of Ca(2+)-ATPase increases [Ca2+]i and causes vasoconstriction. The vasoconstriction produced by thapsigargin is not significantly different between SHR and WKY.

Effect of Na,K-ATPase inhibition on cytosolic free calcium ions in vascular smooth muscle cells of spontaneously hypertensive and normotensive rats

Objective To investigate the role of Na+-Ca2+ exchange in the regulation of cytosolic free Ca2+ and the pathogenesis of primary hypertension. Method: Cytosolic free Ca2+ ([Ca2+]i) in cultured vascular smooth muscle cells from normotensive and spontaneously hypertensive rats of the Münster strain was measured using the fluorescent dye fura-2 after inhibition of Na+,K+ ATPase by ouabain and after addition of angiotensin II. Results: [Ca2+]i showed a rapid increase together with a depolarization of membrane potential as measured by merocyanine 540. The ouabain-induced increase in [Ca2+]i was blocked in Ca2+-free medium and by nifedipine, but incubation with the inhibitor of the Na+-Ca2+ exchange, NiCl2, did not diminish the effect of ouabain. Likewise, in Na+-free medium the response to ouabain was not suppressed. The angiotensin II-induced changes in [Ca2+]i were diminished in Ca2+-free medium and by nifedipine, but enhanced by NiCl2. Conclusion: The increase in [Ca2+]i after Na+,K+ ATPase inhibition is not due to a modulation of Na+-Ca2+ exchange, but to a Ca2+ influx through Ca2+ channels. Changes in Na+-Ca2+ exchange caused by Na+,K+ ATPase inhibition may not play an important role in vascular smooth muscle cells of spontaneously hypertensive rats.

Angiotensin II responses after protein kinase C activation in vascular smooth muscle cells of spontaneously hypertensive rats

To examine the interaction of protein kinase C (PKC) with agonist-induced calcium fluxes in hypertension, cytosolic free calcium ([Ca2+ ]i) was measured in vascular smooth muscle cells (vSMC) of normotensive and spontaneously hypertensive rats (SHR) after incubation with phorbol,-12 myristate,-13 acetate (PMA) and application of angiotensin II (All). To distinguish between calcium influx through voltage-dependent calcium channels and calcium mobilization from intracellular stores, the calcium agonist Bay K 8644 was used. Resting [Ca2+ ]i was 108.0 ± 10.6 nM (mean ± SEM, n = 25) in normotensive and 102.0 ± 11.4 nM(n = 21) in hypertensive cells. After pretreatment with PMA 10-7 M for 60 min, resting [Ca2+ ]i of normotensive vSMC increased to 145.0 ± 13.8 nM (n = 17) while the resting level of the hypertensive cells decreased to 68.0 ± 2.4 nM (n = 14, p < 0.05 as compared with normotensive cells) in hypertensive vSMC. Maximum increase in [Ca2+ ]i induced with 10 M AH for normotensive and hypertensive vSMC was similar: 230.5 ± 34.4 nM (n = 14) and 212.5 ± 26.7 nM (n = 17). After pretreatment with PMA 10~7 M, the maximum increase in [Ca2+ ]i induced by All in hypertensive cells was limited to 108.0 ± 6.2 nM (p < 0.05 as compared with normotensive cells), whereas the increase in [Ca2+ ]i in normotensive vSMC remained the same as before: 211.5 ± 23.4 nM. After administration of 10-5 M Bay K 8644, [Ca2 + ]; increased by 54.3 ± 12.2 nM (n = 4) and 43.4 ± 17.4 nM (n = 5) in normotensive and hypertensive vSMC, respectively. After preincubation with PMA this effect was reduced to 20.3 ± 8.8 nM in normotensive cells and 29.0 ± 4.6 nM in hypertensive cells. We conclude that in hypertensive vSMC either the feedback inhibition of postreceptor signaling after PKC activation is more pronounced or that higher amounts of PKC can be activated.

Effects of Protein Kinase C Activation on Intracellular Ca2+ Distribution in Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats

Protein kinase C is known to influence contraction in vascular smooth muscle cells by Ca(2+)-dependent and Ca(2+)-independent mechanisms. In the present study, the effect of protein kinase C activation by phorbol 12-myristate 13-acetate on resting cytosolic free Ca2+ and on cellular Ca2+ pools was assessed in cultured rat aortic muscle cells using fura 2. Cellular Ca2+ pools were evaluated with the selective inhibitor of the sarcoplasmic Ca2+ ATPase, thapsigargin. In normotensive vascular smooth muscle cells, protein kinase C activation caused a redistribution of Ca2+ from the thapsigargin-sensitive pool into the cytoplasm, whereas, in hypertensive cells, no significant effect of protein kinase C activity on cellular Ca2+ distribution was found. It is concluded that protein kinase C modulates the amount of Ca2+ stored in the thapsigargin-sensitive calcium stores. In hypertensive cells, the regulation of Ca2+ pools by protein kinase C is disturbed.

Mechanism of the Action of Angiotensin-Converting Enzyme Inhibitors on Agonist-Induced Ca2+ Influx

To evaluate the direct effects of the angiotensin-converting enzyme (ACE) inhibitors, captopril, enalaprilat, enalapril (a prodrug without therapeutically significant ACE inhibitory effect) and ramiprilat, on cellular calcium metabolism, the cytosolic free calcium concentration was measured in cultured rat vascular smooth muscle cells using the fluorescent dye, fura-2. Preincubation with captopril, enalaprilat, enalapril, or ramiprilat for 40 min significantly reduced the angiotensin II-induced transplasma membrane calcium influx but did not influence the angiotension II-induced calcium release from internal stores. Captopril and ramiprilat also inhibited arginine vasopressin, but not the thapsigargin-, norepinephrine-, or the BayK 8644-induced changes in cytosolic calcium. Phorbol 12-myristate 13-acetate pretreatment for 30 s caused an increase in the angiotensin II-induced rise in cytosolic calcium. Although both captopril and verapamil reduced responses to angiotensin II to similar extents, only verapamil blocked the ability of phorbol 12-myristate 13-acetate to enhance responses to angiotensin II. It is concluded that ACE inhibitors modulate the effects of some but not all agonist-induced transplasma membrane calcium influx.