Effects of inhibition of the Na+/K+/2Cl− cotransporter on myogenic and angiotensin II responses of the rat afferent arteriole

2007 ◽  
Vol 292 (3) ◽  
pp. F999-F1006 ◽  
Author(s):  
Xuemei Wang ◽  
Jennifer Breaks ◽  
Kathy Loutzenhiser ◽  
Rodger Loutzenhiser
Keyword(s):  
Smooth Muscle ◽  
Time Course ◽  
Rat Kidney ◽  
Tubuloglomerular Feedback ◽  
Sodium Reabsorption ◽  
Dependent Manner ◽  
Afferent Arteriole ◽  
Concentration Dependent Manner ◽  
Vasomotor Function ◽  
Myogenic Responses

The Na+/K+/2Cl− cotransporter (NKCC) plays diverse roles in the kidney, contributing sodium reabsorption and tubuloglomerular feedback (TGF). However, NKCC is also expressed in smooth muscle and inhibitors of this transporter affect contractility in both vascular and nonvascular smooth muscle. In the present study, we investigated the effects of NKCC inhibitors on vasoconstrictor responses of the renal afferent arteriole using the in vitro perfused hydronephrotic rat kidney. This preparation has no tubules and no TGF, eliminating this potential complication. Furosemide and bumetanide inhibited myogenic responses in a concentration-dependent manner. Bumetanide was ∼20-fold more potent (IC50 1.0 vs. 20 μmol/l). At 100 and 10 μmol/l, furosemide and bumetanide inhibited myogenic responses by 72 ± 4 and 68 ± 5%, respectively. The maximal level of inhibition by bumetanide was not affected by nitric oxide synthase inhibition (100 μmol/l NG-nitro-l-arginine methyl ester). However, the time course for the dilation was slowed (from t1/2 = 4.0 ± 0.5 to 8.3 ± 1.7 min, P = 0.04), suggesting either a partial involvement of NO or a permissive effect of NO on relaxation kinetics. Bumetanide also inhibited ANG II-induced afferent arteriolar vasconstriction at similar concentrations. Finally, NKCC1, but not NKCC2, expression was demonstrated in the afferent arteriole by RT-PCR and the presence of NKCC1 in afferent arteriolar myocytes was confirmed by immunohistochemistry. In concert, these results indicate that NKCC modulation is capable of altering myogenic responses by a mechanism that does not involve TGF and suggest a potential role of NKCC1 in the regulation of vasomotor function in the renal microvasculature.

Regulation of KCa-channel activity by cyclic ADP-ribose and ADP-ribose in coronary arterial smooth muscle

1998 ◽  
Vol 275 (3) ◽  
pp. H1002-H1010 ◽  
Author(s):  
Pin-Lan Li ◽  
Ai-Ping Zou ◽  
William B. Campbell
Keyword(s):  
Smooth Muscle ◽  
Time Course ◽  
Control Level ◽  
Internal Surface ◽  
Threshold Concentration ◽  
Dependent Manner ◽  
Channel Activity ◽  
Arterial Smooth Muscle ◽  
Concentration Dependent Manner ◽  
Cyclic Adp Ribose

The enzymatic pathway responsible for the production and metabolism of cyclic ADP-ribose (cADP-R) in small bovine coronary arteries was characterized, and the role of cADP-R and ADP-ribose (ADP-R) in the regulation of the activity of large-conductance Ca2+-activated K+(KCa) channels was determined in vascular smooth muscle cells (SMC) prepared from these vessels. We found that cADP-R and ADP-R were produced when the coronary arterial homogenates were incubated with 1 mM β-NAD. The time course of the enzyme reactions showed that the maximal conversion rate (1.37 ± 0.03 nmol ⋅ min−1 ⋅ mg protein−1) of β-NAD to cADP-R was reached after 3 min of incubation. As incubation time was prolonged, the production of ADP-R was increased to a maximal rate of 3.66 ± 0.03 nmol ⋅ min−1 ⋅ mg protein−1, whereas cADP-R production decreased. Incubation of the homogenate with cADP-R produced a time-dependent increase in the synthesis of ADP-R. Comparison of coronary arterial microsomes with cytosols shows that the production of both cADP-R and ADP-R in microsomes was significantly greater. In excised inside-out membrane patches of single coronary SMC, the KCa channels were activated when β-NAD, the precursor for both cADP-R and ADP-R, was applied to the internal surface. This effect of β-NAD may be associated with the production of ADP-R, because the KCa-channel activity was increased by ADP-R in a concentration-dependent manner. The open-state probability of the KCa channels increased from a control level of 0.08 ± 0.03 to 0.17 ± 0.05 even at the lowest ADP-R concentration (0.1 μM) studied. However, cADP-R reduced the KCa-channel activity, and the threshold concentration of cADP-R that decreased the average channel activity of the KCa channels was 1 μM. These results provide evidence that cADP-R is produced and metabolized in the coronary arterial smooth muscle and that a cADP-R/ADP-R pathway participates in the control of the KCa-channel activity in vascular SMC.

Abstract 36: Deficiency in the Production of 20-HETE Contributes to Impaired Myogenic and TGF Responses in the Afferent Arteriole of Dahl S Rats

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Ying Ge ◽  
Fan Fan ◽  
Sydney R Murphy ◽  
Jan Michael Williams ◽  
Ruisheng Liu ◽  
...  
Keyword(s):  
Renal Injury ◽  
Tubuloglomerular Feedback ◽  
Brown Norway ◽  
Sodium Reabsorption ◽  
Thick Ascending Limb ◽  
Afferent Arteriole ◽  
Synthesis Inhibitor ◽  
Renal Vasculature ◽  
Luminal Diameter ◽  
Consomic Strain

Previous studies have indicated that a deficiency in the formation of 20-HETE in the proximal tubule and thick ascending limb of Henle in Dahl S rats increases sodium reabsorption and contributes to the development of hypertension. The present study examined whether the lack of 20-HETE production in the renal vasculature contributes to the progression of renal injury by altering the myogenic or tubuloglomerular feedback (TGF) response of the afferent arteriole (Af-Art). The production of 20-HETE was significantly lower by 54% in renal microvessels isolated from the kidneys of Dahl S rats versus that seen than in SS.5BN consomic strain in which chromosome 5 from the Brown Norway (BN) rat containing the CYP4A genes responsible for the formation of 20-HETE was transferred into the Dahl S genetic background. The luminal diameter of the Af-Art decreased by 14.7± 1.5% (from 20.5 ± 0.7 to 17.5 ± 0.8 μm, n=6) in SS.5BN rats whereas the diameter of the Af-Art remained unaltered in Dahl S rats (from 20.1 ± 0.6 to 21.7 ± 0.6 μm, n=7) when the perfusion pressure was increased from 60 mmHg to 120 mmHg. In other experiments, adenosine (1 μM) reduced the diameter of the Af-Art in the SS.5BN rats by 15±0.7% (from 20.1 ±0.4 to 17.1 ± 0.9 μm, n=3) whereas the Af-Art of Dahl S rats was unaltered. However, administration of a 20-HETE synthesis inhibitor, HET0016 (1 μM, n=6), or a selective 20-HETE antagonist, 6, 15-20-HEDE (10 μM, n=6) completely blocked the myogenic and adenosine responses in the Af-Art of SS.5BN rats but it had no effect in Dahl S rats. Administration of a 20-HETE agonist, 5, 14-20-HEDE (1 μM) restored the myogenic response (from 20.7 ± 0.7 to 17.6 ± 0.6 μm, n=7) and vasoconstrictor response to adenosine in the Af-Art of Dahl S rats. These studies confirm the key role of 20-HETE in modulating the responsiveness of the Af-Art and indicate that a deficiency in the formation of 20-HETE in renal microvessels contributes to the marked susceptibility of Dahl S rats to develop hypertension induced renal injury.

Properties of neuroprotective cell-permeant Ca2+ chelators: effects on [Ca2+]i and glutamate neurotoxicity in vitro

1994 ◽  
Vol 72 (4) ◽  
pp. 1973-1992 ◽  
Author(s):  
M. Tymianski ◽  
M. P. Charlton ◽  
P. L. Carlen ◽  
C. H. Tator
Keyword(s):  
Time Course ◽  
Dependent Manner ◽  
Protective Effects ◽  
Tetraacetic Acid ◽  
Acetoxymethyl Ester ◽  
Neuroprotective Effects ◽  
Concentration Dependent Manner ◽  
Fura 2 ◽  
Glutamate Neurotoxicity

1. Cell-permeant Ca2+ chelators such as 1,2-bis-(2-amino-phenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) protect neurons against excitotoxic and ischemic neuronal injury in vitro and in vivo. Here we provide the first steps toward characterizing the mechanisms by which these agents produce their neuroprotective effects. 2. Cultured mouse spinal neurons were simultaneously loaded with the Ca2+ indicator fura-2 and with one of three permeant chelators derived from the fast Ca2+ buffer BAPTA, or with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Adding these chelators did not interfere with the fluorescence spectrum of fura-2 and had no effect on baseline [Ca2+]i. 3. The neurons were challenged with 250 microM L-glutamate for 50 min, producing a marked transient [Ca2+]i increase followed by a decay of [Ca2+]i to a lower “plateau.” About 80% of control neurons succumbed to this excitotoxic insult. Neurons that survived adjusted their plateau [Ca2+]i to lower levels than those that succumbed. 4. Neurons that were pretreated with permeant Ca2+ chelators became more resistant to these neurotoxic challenges. 5. We examined whether this reduction in glutamate neurotoxicity could be related to the given buffer's known Ca2+ affinity (Kd), its Ca2+ binding kinetics, and its ability to attenuate glutamate-induced [Ca2+]i increases. 6. Pretreatment of neurons with BAPTA analogues having Kds ranging from 100 to 3,600 microM 1) attenuated the amplitude and 2) lengthened the time constant describing the rise and decay of the glutamate-evoked [Ca2+]i transient. The magnitude of these effects paralleled the affinity of the chelator for Ca2+. 7. BAPTA-AM and its analogues dramatically attenuated the early neurotoxicity of glutamate, reducing cell deaths by up to 80%. However, in contrast with the graded effects of chelators having different Ca2+ affinities on Ca2+ transients, all BAPTA analogues were equally protective. These protective effects did not relate to the chelators' Ca2+ affinity within a Kd range of 100 nM (for BAPTA) to 3,600 nM (for 5,5'-dibromo BAPTA). 8. BAPTA-AM protected neurons in a concentration-dependent manner with 50% protection obtained with 10 microM, a concentration having no effect on the [Ca2+]i transient amplitude. 9. EGTA, a slow Ca2+ buffer with a similar Ca2+ affinity to BAPTA produced the same effects as BAPTA on [Ca2+]i transient kinetics. However, it was far less protective than BAPTA. 10. The time course of early glutamate neurotoxicity was altered by the BAPTA analogues, but not EGTA. BAPTA analogues caused a small increase in cell deaths in the first minutes of each experiment, followed by relative sparing from further neurodegeneration. 11. The ability of low Ca2+ affinity chelators such as 5,5'-dibromo BAPTA to protect neurons without markedly attenuating measured [Ca2+]i increases conflicts with the hypothesis that global elevations in [Ca2+]i are responsible for triggering neurotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Sevoflurane Inhibits Guanosine 5′-[γ-thio]triphosphate–stimulated, Rho/Rho-kinase–mediated Contraction of Isolated Rat Aortic Smooth Muscle

2003 ◽  
Vol 99 (3) ◽  
pp. 646-651 ◽  
Author(s):  
Jingui Yu ◽  
Koji Ogawa ◽  
Yasuyuki Tokinaga ◽  
Yoshio Hatano
Keyword(s):  
Smooth Muscle ◽  
Kinase Inhibitor ◽  
Isometric Force ◽  
Rho Kinase ◽  
Force Transducer ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Aortic Smooth Muscle ◽  
Gamma S ◽  
Kinase Pathway

Background The Rho/Rho-kinase signaling pathway plays an important role in mediating Ca2+ sensitization of vascular smooth muscle. The effect of anesthetics on Rho/Rho-kinase-mediated vasoconstriction has not been determined to date. This study is designed to examine the possible inhibitory effects of sevoflurane on the Rho/Rho-kinase pathway by measuring guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)-stimulated contraction and translocation of RhoA (one of the three Rho subtypes) and Rock-2 (one of the two Rho-kinase subtypes) from the cytosol to the membrane in rat aortic smooth muscle. Methods GTP gamma S-induced contraction of rat aortic endothelium-denuded rings was measured using an isometric force transducer, and GTP gamma S-stimulated membrane translocation of RhoA and Rock-2 in smooth muscle cells was detected with Western blotting in the presence and absence of sevoflurane. Results GTP gamma S (10(-4) m) induced a sustained contraction, which was significantly inhibited by the Rho-kinase inhibitor, Y27632 (3 x 10(-6) m). Before treatment with GTP gamma S, RhoA and Rock-2 were detected primarily in the cytosolic fraction. GTP gamma S (10(-4) m) stimulated the translocation of RhoA and Rock-2 from the cytosol to the membrane, which was sustained for more than 60 min. Sevoflurane (1.7, 3.4, and 5.1%) concentration dependently inhibited the GTP gamma S-induced constriction of rat aortic smooth muscle with a reduction of constriction of 52-75% (P < 0.01, n = 8), and attenuated the translocation of RhoA and Rock-2 by 31-66% and 34-78%, respectively (P < 0.05-0.01, respectively; n = 4). Conclusion The current findings show that sevoflurane depresses the GTP gamma S-stimulated contraction and translocation of both Rho and Rho-kinase from the cytosol in a concentration-dependent manner, indicating that sevoflurane is able to inhibit vasoconstriction mediated by the Rho/Rho-kinase pathway in rat aortic smooth muscle.

scholarly journals Renal microvascular actions of calcitonin gene-related peptide

1998 ◽  
Vol 274 (6) ◽  
pp. F1078-F1085 ◽  
Author(s):  
Martina Reslerova ◽  
Rodger Loutzenhiser
Keyword(s):  
Angiotensin Ii ◽  
Rat Kidney ◽  
Elevated Pressure ◽  
Calcitonin Gene Related Peptide ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Renal Vasoconstriction ◽  
Related Peptide ◽  
Calcitonin Gene

Calcitonin gene-related peptide (CGRP) is a potent vasodilator that is suggested to act via ATP-sensitive K channels (KATP). In the present study, we examined the actions of CGRP on pressure- and angiotensin II-induced vasoconstriction, using the in vitro perfused hydronephrotic rat kidney. Elevated pressure (from 80 to 180 mmHg) and 0.1 nM angiotensin II elicited similar decreases in afferent diameter in this model. CGRP inhibited myogenic reactivity in a concentration-dependent manner, completely preventing pressure-induced constriction at 10 nM (95 ± 10% inhibition). These effects were partially attenuated by 10 μM glibenclamide (62 ± 16% inhibition, P = 0.025), indicating both KATP-dependent and -independent actions of CGRP. In contrast, 10 nM CGRP inhibited angiotensin II-induced vasoconstriction by only 54 ± 11%, and this action was not affected by glibenclamide (41 ± 11%, P = 0.31). CGRP also inhibited the efferent arteriolar response to angiotensin II in the absence and presence of glibenclamide. Pinacidil (1.0 μM), a KATP opener also preferentially inhibited pressure- vs. angiotensin II-induced vasoconstriction (97 ± 5 and 59 ± 13% inhibition, respectively; P = 0.034). We conclude that the renal vasodilatory mechanisms of CGRP are pleiotropic and involve both KATP-dependent and -independent pathways. The effectiveness of CGRP in opposing renal vasoconstriction and the role of KATP in this action appear to depend on the nature the underlying vasoconstriction. We suggest that this phenomenon reflects an inhibition of KATP activation by angiotensin II.

Differential blockade of agonist- and depolarization-induced 45Ca2+ influx in smooth muscle cells

1989 ◽  
Vol 257 (4) ◽  
pp. C607-C611 ◽  
Author(s):  
A. Wallnofer ◽  
C. Cauvin ◽  
T. W. Lategan ◽  
U. T. Ruegg
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Gamma S ◽  
Ca2 Channels ◽  
Stimulation Of

ATP stimulated 45Ca2+ influx in rat aortic smooth muscle cells in a concentration-dependent manner (EC50 = 3.6 +/- 0.5 X 10(-7) M). ADP and GTP were less effective than ATP in stimulating 45Ca2+ influx; AMP was weakly active and the adenosine agonist 5'-(N-ethyl-carboxamido)-adenosine (NECA) had no effect. ATP gamma S was about equieffective with ATP, whereas alpha,beta-methylene-ATP (APCPP) did not induce 45Ca2+ influx. Stimulation of 45Ca2+ influx by ATP was not abolished by the dihydropyridine Ca2+ channel antagonist darodipine (PY 108-068), which completely blocked depolarization-induced 45Ca2+ influx. Inorganic cations (La3+, Cd2+, Co2+, Ni2+, Mn2+, and Mg2+) were able to inhibit both agonist- and depolarization-induced 45Ca2+ influx. Cd2+, however, was approximately 20 times more selective in blocking K+-stimulated than agonist-stimulated 45Ca2+ influx. These data indicate that ATP-stimulated Ca2+ influx in rat aortic smooth muscle cells is resistant to darodipine but is reduced by La3+, Cd2+, and other inorganic blockers of Ca2+ channels.

Control of neurotransmission by prostaglandins in canine trachealis smooth muscle

1984 ◽  
Vol 57 (1) ◽  
pp. 129-134 ◽  
Author(s):  
E. H. Walters ◽  
P. M. O'Byrne ◽  
L. M. Fabbri ◽  
P. D. Graf ◽  
M. J. Holtzman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Prostaglandin E2 ◽  
Electrical Field Stimulation ◽  
Tracheal Smooth Muscle ◽  
Dependent Manner ◽  
Field Stimulation ◽  
Electrical Field ◽  
Similar Time ◽  
Concentration Dependent Manner ◽  
Contractile Responses

Contractile responses of canine tracheal smooth muscle to electrical field stimulation diminished over a 2-h period of incubation. However, addition of indomethacin (10(-5) M) for a similar time not only prevented this inhibition of contractile response, but actually markedly increased the response to electrical field stimulation, suggesting that prostaglandins were responsible for the time-dependent inhibition. Measured prostaglandin E2 increased in the tissue bath over 2 h in control tissues. Addition of prostaglandin E2 to the tissue produced similar inhibition of contractile responses to electrical field stimulation in a concentration-dependent manner. In contrast, incubation alone, treatment with indomethacin, or addition of prostaglandin E2 had little, if any, effect on contractions induced by acetylcholine. We conclude that the release of prostaglandins from canine tracheal smooth muscle that occurs with time has a predominantly inhibitory effect on cholinergic neurotransmission at a prejunctional site.

Neural and myogenic effects of cyclooxygenase products on canine bronchial smooth muscle

1995 ◽  
Vol 268 (1) ◽  
pp. L47-L55 ◽  
Author(s):  
A. P. Abela ◽  
E. E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Electrical Field Stimulation ◽  
Resting Membrane Potential ◽  
Dependent Manner ◽  
Ach Release ◽  
Concentration Dependent Manner ◽  
Excitatory Junction Potentials ◽  
Pg E2 ◽  
Large Contraction

In canine bronchi bathed in 10(-6) M indomethacin (IDM), prostaglandin (PG) E2 inhibited electrical field stimulation (EFS)- and acetylcholine (ACh)-mediated contractions and excitatory junction potentials (EJP) in a concentration-dependent manner without altering the resting membrane potential. EFS-induced EJPs were abolished at 10(-7) M PGE2, which shifted responses to ACh 10-fold rightward. Thus PGE2 predominantly inhibited the release of ACh and secondarily decreased smooth muscle response to ACh. U-46619, an analogue of thromboxane A2 (TxA2), initiated tetrodotoxin- and atropine-insensitive contractions in a concentration-dependent manner. U-46619 (10(-9) M) did not alter significantly EFS- or ACh-stimulated contractions and potentiated EFS amplitude of EJPs without depolarizing muscle cells. Either prejunctional activation of ACh release by TxA2 or postjunctional potentiation of the response to ACh can explain these findings. U-46619 (<or = 10(-8) M) depolarized the membrane potential, initiating oscillations accompanied by a large contraction. Addition of 10(-8) M nitrendipine, but not tetraethylammonium (25 mM), blocked the oscillations selectively. Other prostanoids (PGD2, PGI2, and PGF2 alpha) had no significant effects on canine bronchi. In the absence of IDM, PGE2 accumulated, EFS contractions decreased with time, and EJPs disappeared. We conclude that in canine bronchi PGE2 predominantly inhibits ACh release and endogenous PGE2 acts similarly, whereas TxA2 excites, probably at postjunctional sites.

Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells

2002 ◽  
Vol 282 (2) ◽  
pp. G226-G232 ◽  
Author(s):  
Hirotada Akiho ◽  
Patricia Blennerhassett ◽  
Yikang Deng ◽  
Stephen M. Collins
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Trichinella Spiralis ◽  
Muscle Cells ◽  
Dependent Manner ◽  
T Helper ◽  
Concentration Dependent Manner ◽  
T Helper 2 ◽  
Muscularis Externa

T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13, which activate signal transducer and activator of transcription 6 (STAT6) are expressed in the muscularis externa during nematode infection and are candidate mediators of the associated hypercontractility. To determine the locus of action of these cytokines, we examined the IL-4- and IL-13-induced hypercontractility of the isolated muscle cells from STAT6 +/+ and STAT6 −/− mice. We compared the results with cells isolated from Trichinella spiralis-infected STAT6 +/+ and STAT6 −/− mice. Carbamylcholine chloride (Carbachol) induced the contraction of jejunal muscle cells in a concentration-dependent manner maximal contraction (Rmax26.7 ± 1.9%). Cells from T. spiralis-infected STAT6 −/− mice showed the hypertrophy (cell lengths 41.4 ± 0.8 to 89.0 ± 8.7 μm) and hypercontractility (Rmax37.5 ± 1.3%) induced by infection. IL-4Rα mRNA was detected in dispersed smooth muscle cells. Incubation of longitudinal muscle-myenteric plexus (LMMP) with IL-4 and IL-13 enhanced Carbachol-induced muscle contraction (Rmax35.5 ± 1.9 and 32.4 ± 2.9%, respectively). Incubation of LMMP from STAT6 −/− mice with IL-4 did not enhance the contraction. The hypercontractility in T. spiralis-infected mice was attenuated in STAT6 −/− mice ( P < 0.02). These results indicate both IL-4 and IL-13 induce hypercontractility of muscle cells via the STAT6 pathway, and this is the basis for hypercontractility observed in T. spiralis-infected mice.

scholarly journals The contribution of inositol 1,4,5-trisphosphate and ryanodine receptors to agonist-induced Ca2+ signaling of airway smooth muscle cells

2009 ◽  
Vol 297 (2) ◽  
pp. L347-L361 ◽  
Author(s):  
Yan Bai ◽  
Martin Edelmann ◽  
Michael J. Sanderson
Keyword(s):  
Wave Propagation ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Inositol 1,4,5 Trisphosphate

The relative contribution of inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs) to agonist-induced Ca2+ signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phase-contrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptamine (5-HT) induced Ca2+ oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an IP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the Ca2+ oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited Ca2+ waves generated by the photolytic release of IP3. However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or IP3-induced Ca2+ oscillations or Ca2+ wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced Ca2+ oscillations in a concentration-dependent manner. However, tetracaine did not affect IP3-induced Ca2+ release or wave propagation nor the Ca2+ content of SMC Ca2+ stores as evaluated by Ca2+-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow Ca2+ oscillations induced by KCl. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or Ca2+ oscillations of SMCs were also observed at 37°C. In Ca2+-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular Ca2+ levels indicating that relaxation also resulted from a reduction in Ca2+ sensitivity. These results indicate that agonist-induced Ca2+ oscillations in mouse small airway SMCs are primary mediated via IP3Rs and that tetracaine induces relaxation by both decreasing Ca2+ sensitivity and inhibiting agonist-induced Ca2+ oscillations via an IP3-dependent mechanism.

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