Effects of serotonin on intracellular pH and contraction in vascular smooth muscle.

Abstract

Serotonin (5-HT) and other contractile agonists stimulate Na(+)-H+ exchange in vascular smooth muscle. Since intracellular alkalinization, per se, stimulates contraction, we tested whether 5-HT-induced contraction was associated with an increased pHi. In HCO3(-)-free buffer (pHo 7.4), 5-HT (10(-5) M) increased pHi, as measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence, from 7.10 +/- 0.03 to 7.34 +/- 0.03 (p < 0.01) in primary cultures of canine femoral artery vascular smooth muscle cells grown to confluence in the presence of 10% fetal calf serum. In HCO3- buffer (24 mM, pHo 7.4), resting pHi was 7.26 +/- 0.04 (p < 0.01 versus HCO3(-)-free buffer) but was not altered by 5-HT. In both types of buffer, 5-HT stimulated 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake (Na(+)-H+ exchange). In HCO3- buffer and in Na(+)- and HCO3(-)-free buffer, 5-HT increased 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- uptake, suggesting that 5-HT stimulated Na(+)-independent Cl(-)-HCO3- and Cl(-)-Cl- exchange activities, respectively. Individual vascular smooth muscle cells were then cultured on rat tail tendon collagen gels in the presence of 0.5% fetal calf serum, and cell length and pHi were measured by video and epifluorescence microscopy. 5-HT contracted cells in a dose-dependent, reversible, and ketanserin-inhibitable manner. These cells, like cells grown in 10% fetal calf serum, exhibited Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange. In HCO3- buffer, 5-HT contracted cells without an associated change in pHi. We concluded the following: 1) 5-HT stimulated both Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange activities in cultured vascular smooth muscle cells in parallel. 2) As a result of enhanced H+ and HCO3- efflux, pHi was not altered. 3) In the presence of HCO3-, 5-HT-induced contraction was not associated with a change in pHi.

Evidence for a carotid body homolog in the lizardTupinambis merianae

“…We predicted that we would see an increase in MAP upon injection of 5-HT and norepinephrine because both of these neurotransmitters are known to cause vasoconstriction (Vanhoutte, 1974;Kahn et al, 1992) whereas ACh does not (Kirby and Burnstock, 1969;Vanhoutte, 1974). Our data match these predictions.…”

Insulin reduces contraction and intracellular calcium concentration in vascular smooth muscle.

“…8 We have previously shown that the cells in this preparation are contracted by serotonin in a dose-dependent, inhibitable, and reversible manner. 8 The dishes were placed on the heated (37°C) stage of a Nikon Diaphot inverted phase-contrast microscope, and the culture medium was replaced with a physiological salt solution that contained (mmol/L) NaCl, 140; KC1, 4; CaCl 2 , 2; MgCl 2 , 1; glucose, 5; and HEPES-Tris, 10, pH 7.4 (HEPES PSS) plus 4% bovine serum albumen (BSA), with or without bovine insulin (40 juU/mL) (Sigma) (Figs 2 through 4 and 6 through 8). After 20 minutes, a field of at least 6 to 10 cells was photographed at x200 to obtain baseline images.…”

“…The dispersed cells were pelleted and washed three times in Hanks' balanced salt solution (GIBCO, Grand Island, NY) and suspended to a density of 2X10 3 cells per milliliter in Dulbecco's modified Eagle's medium (DMEM) that contained 0.5% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin solution (10 000 U/mL penicillin, 10 mg/mL streptomycin, Sigma). One milliliter of this suspension was placed in 35-mm culture dishes on top of rat tail tendon collagen gels, which were prepared as follows 8 : Sprague-Dawley rat tail tendons were sterilized in 70% ethanol for 4 hours, minced, and extracted with 0.1% acetic acid for 48 hours at 4°C. The protein concentration of the supernatant was adjusted to 0.15 mg/mL and titrated to pH 8.0 with NaOH at 4°C.…”

Mitogen-Activated Protein/Extracellular Signal–Regulated Kinase Inhibition Attenuates Angiotensin II–Mediated Signaling and Contraction in Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells

“…Contraction was measured on the basis of the methods described by Kahn et al 27 and Bodin et al 28 Cells were loaded with fura-2-acetoxymethyl ester (4 mol/L) for [Ca 2ϩ ] i or BCECF (0.2 mol/L) for pH i , as described in detail previously. 16,29 Single cells in cell clusters were investigated using an Axiovert 135 inverted microscope (ϫ40 oil-immersion objective) and an Attofluor digital fluorescence system (Zeiss) using alternating excitatory wavelengths of 343 and 380 nm for [Ca 2ϩ ] i measurements and 440 and 495 nm for pH i measurements.…”

“…The magnitude of cell contraction was expressed as the percentage reduction in cell length relative to initial baseline measurements. 16,27,28 For each cell, the percentage contraction from the baseline was calculated, and these values were averaged for all cells. The average basal cell measurements were consistent between preparations.…”