Affiliation:
1. From the Experimental Hypertension Laboratory, MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal and Université de Montréal, Montreal, Quebec, Canada.
Abstract
Abstract
Intracellular Ca
2+
and pH are potent modulators of growth factor–induced mitogenesis and contraction. This study examined platelet-derived growth factor–(PDGF-BB) and insulin-like growth factor (IGF-1)–mediated signal transduction in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric arteries of Sprague-Dawley rats. Intracellular free Ca
2+
concentration ([Ca
2+
]
i
) and intracellular pH (pH
i
) were measured by fluorescence digital imaging using fura-2 AM and 2′7′-bis(2-carboxyethyl)-5
6
-carboxyfluorescein, respectively. Characteristics of [Ca
2+
]
i
transients were determined by pre-exposing cells to Ca
2+
-free buffer, and involvement of the Na
+
/Ca
2+
exchanger was assessed by withdrawal of extracellular Na
+
and by exposure to dimethylbenzamil (Na
+
/Ca
2+
exchange blocker). To determine whether pH
i
responses were mediated via the Na
+
/H
+
exchanger, cells were preincubated with 10
−5
mol
/L
5-(N-ethyl-N-isopropyl)amiloride (a selective Na
+
/H
+
exchange blocker). The role of protein kinase C (PKC) and tyrosine kinases in growth factor signaling was assessed by pre-exposing cells to calphostin C and chelerythrine chloride (selective PKC inhibitors; 10
−5
mol
/L
) and tyrphostin A23 (a selective tyrosine kinase inhibitor; 10
−5
mol
/L
). PDGF-BB and IGF-1 (1 to 10 ng/mL) increased [Ca
2+
]
i
and pH
i
in a dose-dependent manner. At concentrations greater than 1 ng/mL both growth factors induced a biphasic [Ca
2+
]
i
response with an initial transient peak followed by a sustained elevation. At 5 ng/mL PDGF-BB and IGF-1 significantly increased [Ca
2+
]
i
from 95±3 nmol/L to 328±28 and 251±18 nmol/L, respectively. Ca
2+
withdrawal abolished the second phase of [Ca
2+
]
i
elevation. Agonist-induced [Ca
2+
]
i
responses were similarly altered by Na
+
withdrawal, by Na
+
/Ca
2+
exchange blockade, and by PKC inhibition; latency, the period from stimulus application to the first [Ca
2+
]
i
peak, was increased, the initial [Ca
2+
]
i
peak was attenuated, and the sustained phase was prolonged. PDGF-BB and IGF-1 (10 ng/mL) significantly increased pH
i
from 6.89±0.04 nmol/L to 7.11±0.01 and 7.09±0.02 nmol/L, respectively. EIPA and calphostin C completely inhibited agonist-elicited alkalinization. Tyrphostin A-23 abolished second-messenger responses to PDGF-BB and IGF-1, whose receptors have tyrosine kinase activity. In conclusion, PDGF-BB and IGF-1 elicit significant [Ca
2+
]
i
and pH
i
responses in VSMC. The underlying pathways that mediate these responses are partially dependent on Na
+
/Ca
2+
transporters and the Na
+
/H
+
exchanger, both of which are linked to PKC activation.
Publisher
Ovid Technologies (Wolters Kluwer Health)
Cited by
31 articles.
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