Receptors for substance P on isolated intestinal smooth muscle cells of the guinea pig

1987 ◽  
Vol 253 (5) ◽  
pp. G666-G672
Author(s):  
J. C. Souquet ◽  
K. N. Bitar ◽  
J. R. Grider ◽  
G. M. Makhlouf
Keyword(s):  
Smooth Muscle ◽  
Substance P ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Muscle Cells ◽  
Receptor Subtype ◽  
Longitudinal Muscle Layer ◽  
Substance K

Two radioligands, 125I-labeled substance P (125I-SP) and 125I-labeled substance K (125I-SK), were used to characterize the kinetics and stoichiometry of binding of mammalian tachykinins [substance P (SP), substance K (SK), and neuromedin K (NK)] to smooth muscle cells isolated from the longitudinal muscle layer of guinea pig intestine. Specific binding of 125I-SP and 125I-SK was rapid, saturable, reversible, and temperature dependent. Binding attained 63-70% of steady-state binding within 1 min, coincidentally with the time of optimal contraction. The order of potency with which mammalian tachykinins and the SP antagonist, [D-Pro2, D-Trp7,9]SP, inhibited the binding of both radioligands was identical: SP greater than SK greater than NK greater than [D-Pro2, D-Trp7,9]SP, implying preferential interaction with a site that had highest affinity for SP. SK was 2-3 times, NK 3-4 times, and [D-Pro2, D-Trp7,9]SP 7-23 times less potent than SP (IC50 0.36 nM). Except for NK, the order of potency was similar to that for contraction of isolated muscle cells. The existence of binding sites with even higher affinity was suggested by the ability of muscle cells to contract in response to concentrations as low as 10(-13) M. These binding sites were not detectable at the concentration of radioligands used. It was concluded that a SP receptor is the only tachykinin receptor subtype present on intestinal muscle cells of the guinea pig.

Receptors for mammalian tachykinins on isolated intestinal smooth muscle cells

1985 ◽  
Vol 249 (4) ◽  
pp. G533-G538
Author(s):  
J. C. Souquet ◽  
J. R. Grider ◽  
K. N. Bitar ◽  
G. M. Makhlouf
Keyword(s):  
Smooth Muscle ◽  
Substance P ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Rank Order ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Receptor Subtype ◽  
Response Curves ◽  
Isolated Cells

The existence of receptors for three mammalian tachykinins, substance P (SP), substance K (SK), and neuromedin K (NK), was examined in smooth muscle cells, isolated separately from the longitudinal and circular muscle layers of guinea pig ileum. Tachykinin receptors capable of mediating contraction were present in muscle cells from both layers. The receptors were selectively blocked by the tachykinin antagonist [D-Pro2, D-Trp7,9]substance P but not by muscarinic, gastrin/cholecystokinin, or opiate antagonists (0.3 nM atropine, 1 mM proglumide, and 0.3 nM naloxone, respectively). The rank order of potency of tachykinins in causing contraction, NK greater than SP greater than SK, was similar in both muscle cell types. The results obtained in isolated muscle cells were closely paralleled by results obtained in intact muscle strips; the main difference was the greater sensitivity of isolated cells to tachykinin agonists (250-fold) and antagonist (210-fold). The inhibitory dissociation constant (Ki) of [D-Pro2, D-Trp7,9]substance P estimated from the displacement of dose-response curves (muscle cells) or from Schild plots (muscle strips) differed minimally or not at all, when either SP or SK was used as agonist, consistent with interaction of the two peptides with the same receptor subtype. The notion of a single receptor subtype in ileal muscle cells of the guinea pig was further supported by the occurrence of complete cross-desensitization between SP and SK in muscle strips.

Dissociation of contraction and muscarinic receptor binding to isolated smooth muscle cells

1986 ◽  
Vol 251 (4) ◽  
pp. G546-G552 ◽  
Author(s):  
S. M. Collins ◽  
D. J. Crankshaw
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Muscle Cells ◽  
Muscarinic Agonists ◽  
Isolated Cells ◽  
Single Class ◽  
Antagonist Binding ◽  
Isolated Smooth Muscle Cells

We examined changes in [3H]QNB binding and cell length induced by muscarinic ligands in a suspension of single smooth muscle cells isolated from the canine stomach. Cells contracted following a brief (30 s) exposure to picomolar concentrations of muscarinic agonists and yielded ED50 values of 1.0 +/- 0.7 pM for oxotremorine, 12.5 +/- 1.8 pM for carbachol, and 16.0 +/- 2.9 pM for metacholine. Contraction was inhibited by atropine with a pA2 value of 10.2 +/- 1.1. The binding of [3H]QNB was rapid and reversible and was stereospecific and pharmacologically appropriate. Specific binding of [3H]QNB was saturable and bound with high affinity (KD 1.04 +/- 0.23 nM) to a single class of sites, of which there were approximately 200,000/cell. In competition experiments antagonist binding was generally homogeneous, whereas that of agonists was heterogeneous and subpopulations of binding sites with different affinities for agonists were identified. The Ki value of 8.1 +/- 1.1 nM for inhibition of QNB binding by atropine was greater than the pA2 of 10.2 +/- 1.1 derived from contraction studies. Furthermore, whereas picomolar concentrations of agonists induced cell contraction, substantially higher concentrations (10 nM to 10 mM) were required to inhibit [3H]QNB binding to the isolated cells.

Evidence for the existence of F2-isoprostane receptors on rat vascular smooth muscle cells

1993 ◽  
Vol 264 (6) ◽  
pp. C1619-C1624 ◽  
Author(s):  
M. Fukunaga ◽  
N. Makita ◽  
L. J. Roberts ◽  
J. D. Morrow ◽  
K. Takahashi ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Tissue Injury ◽  
Specific Binding ◽  
Muscle Cells ◽  
Free Oxygen Radicals ◽  
Txa2 Receptor

The isoprostanes are nonenzymatically generated prostanoids synthesized in vivo in humans and rats through reactions catalyzed by free oxygen radicals. 8-Epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), an F2-isoprostane, is a potent smooth muscle constrictor. A thromboxane A2 (TxA2) receptor antagonist, SQ 29548, blocks renal vasoconstriction during 8-epi-PGF2 alpha administration in rats. With the use of cultured rat aortic smooth muscle cells, we found specific binding sites for [3H]SQ 29548 and for [125I]BOP, a TxA2 agonist. Both ligands were displaced from these binding sites by 8-epi-PGF2 alpha, although with significantly lesser potency than nonlabeled SQ 29548, I-BOP, or U-46619, a TxA2 agonist. In contrast, 8-epi-PGF2 alpha stimulated inositol 1,4,5-trisphosphate production and DNA synthesis in these cells with significantly greater potency than any TxA2 agonist, effects only partially inhibited by SQ 29548. In human TxA2 receptor cDNA-transfected cells, competition by 8-epi-PGF2 alpha for specific [3H]SQ 29548 binding was negligible. Thus 8-epi-PGF2 alpha probably exerts its biological actions in vascular smooth muscle through activation of receptor sites related to but distinct from TxA2 receptors. The existence of such binding sites suggests novel avenues for investigation into the biology of TxA2 and of free radical-mediated tissue injury.

Vasopressin and angiotensin II receptors in rat aortic smooth muscle cells in culture

1983 ◽  
Vol 244 (1) ◽  
pp. E72-E82 ◽  
Author(s):  
J. Penit ◽  
M. Faure ◽  
S. Jard
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Muscle Cells ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Specific Binding Sites

Rat aortic smooth muscle cells were isolated and maintained in primary culture. After 2-3 days, cells recovered their contractile phenotype and could be induced to contract in response to vasopressin and angiotensin II. Vasopressin- and angiotensin-specific binding sites were detected on these cells, using tritiated Lys8-vasopressin, Asn1-Val5-angiotensin II, and Sarc1-Ile8-angiotensin II. Vasopressin binding sites had Kd values of 30 and 12 nM for Lys8-and Arg8-vasopressin, respectively, and a maximal binding capacity of 25,000 sites/cell. They displayed several of the expected characteristics of vasopressin receptors involved in the vasopressor response in vivo. A highly significant correlation was found between the relative agonistic or antagonistic vasopressor potencies of a series of vasopressin structural analogues and their relative abilities to inhibit [3H]vasopressin binding to aortic smooth muscle cells. Specific binding sites for Asn1-Val5-angiotensin II and Sarc1-Ile8-angiotensin II had the following characteristics: Kd = 2.3 and 1.3 nM, respectively; maximal capacity: 50,000 sites/cell. Vasopressin and angiotensin did not modify the intracellular cyclic AMP content of aortic smooth muscle cells.

Substance P activates Cl− and K+ conductances in guinea-pig tracheal smooth muscle cells

1994 ◽  
Vol 72 (6) ◽  
pp. 705-710 ◽  
Author(s):  
Luke J. Janssen ◽  
Stephen M. Sims
Keyword(s):  
Smooth Muscle ◽  
Substance P ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Voltage Clamp ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Equilibrium Potential ◽  
Airway Smooth Muscle Cells ◽  
Inward Current

Substance P (SP) causes bronchoconstriction, but its effects on airway smooth muscle ion conductances are unknown. We investigated the effects of SP on single smooth muscle cells dissociated from guinea-pig trachealis. Under voltage clamp at −60 mV, SP evoked reversible contractions and inward current (ISP). ISP had a latency of approximately 1 s, reached a peak of 1039 ± 147 pA (n = 19) about 2 s after onset of application, and declined to baseline levels over the next 5–10 s. At more positive holding potentials (−25 and 0 mV), the inward current was decreased in magnitude and preceded by outward current. With 140 mM K+ in the electrode and Cl− equilibrium potential (ECl) of about 0 mV, ISP was outwardly rectifying and reversed at −11 ± 2 mV. When K+ currents were blocked using Cs+, the current–voltage relationship for ISP was linear and reversed at 3 ± 1 mV. The reversal potential was dependent on the Cl− gradient across the membrane. These results suggest that SP caused a transient activation of Cl− and K+ conductances. Following the initial transient inward current, SP caused a prolonged suppression of spontaneously active K+ currents. The findings that SP evoked contractions during voltage clamp at potentials at which voltage-dependent Ca2+ channels are not active, and that current oscillations were also evoked by SP, suggest that SP is acting through release of Ca2+ from internal stores. Furthermore, SP occluded the inward current evoked by acetylcholine, suggesting that the peptidergic and cholinergic signalling pathways converge. We conclude that SP releases Ca2+ from internal stores in guinea-pig airway smooth muscle cells, leading to activation of Cl− and K+ conductances, depolarization, and contraction.Key words: Ca2+-dependent conductances, spontaneous transient outward currents, acetylcholine.

Relationship between sensitivity and density of muscarinic receptors in single smooth muscle cells of guinea pig taenia caecum prepared under three conditions

1990 ◽  
Vol 68 (8) ◽  
pp. 1143-1147 ◽  
Author(s):  
Katsuo Koike ◽  
Hiroshi Ohtsuki ◽  
Issei Takayanagi
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Muscarinic Receptors ◽  
Binding Sites ◽  
Single Cells ◽  
Total Concentration ◽  
Muscle Cells ◽  
The Relationship ◽  
Quinuclidinyl Benzilate

The relationship between the sensitivity (the pD2 value) of carbachol and the density (the total concentration of receptors) of muscarinic receptors using single cells from the guinea pig taenia caecum prepared with a mixture of crude collagenase and trypsin inhibitor, purified collagenase alone, and a mixture of purified collagenase and papain was examined. The sensitivity of the single cells prepared with a mixture of purified collagenase and papain was about 10 times more effective than that of the single cells prepared under other conditions. The dissociation constant of [3H]quinuclidinyl benzilate (QNB) and Hill's coefficient did not change in the single cells prepared under the three conditions, though the maximum binding sites were significantly greater in the cells prepared with the mixture of purified collagenase and papain than in those prepared by other means. These results suggest that the increase in the sensitivity of carbachol obtained in the single cells prepared with this mixture is due to the increase in the density of muscarinic receptors and also suggest that the effects of this enzyme mixture may be due to an increase in the incorporation of newly synthesized receptors and (or) changes in receptor turnover.Key words: single smooth muscle cells, muscarinic receptors, sensitivity, density, guinea pig taenia caecum.

Comparison of vasopressin binding sites in human uterine and vascular smooth muscle cells

1999 ◽  
Vol 378 (1) ◽  
pp. 137-142 ◽  
Author(s):  
Atsuo Tahara ◽  
Junko Tsukada ◽  
Noe Ishii ◽  
Yuichi Tomura ◽  
Koh-ichi Wada ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Binding Sites ◽  
Muscle Cells
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