A Nelemans

The inhibitory action of suramin on the P2-purinoceptor response in smooth muscle cells of guinea-pig taenia caeci

The effect of suramin on the smooth muscle cell response of guinea-pig taenia caeci to P2-purinoceptor and alpha 1-adrenoceptor stimulation was measured. The ATP-induced relaxation in potassium (20 mM) pre-contracted taenia caeci was inhibited by suramin (3 x 10(-4) M). The P2-purinoceptor-induced hyperpolarization elicited by ATP both in the presence and absence of calcium was also reduced by suramin. The alpha 1-adrenoceptor-mediated relaxation evoked by phenylephrine was only affected by suramin at low concentrations of the agonist. The results indicate that suramin inhibits the ATP response by interacting with P2y-purinoceptors.

Intracellular Angiotensin II and cell growth of vascular smooth muscle cells

We recently demonstrated that intracellular application of Angiotensin II (Angiotensin IIintr) induces rat aorta contraction independent of plasma membrane Angiotensin II receptors. In this study we investigated the effects of Angiotensin IIintr on cell growth in A7r5 smooth muscle cells. DNA‐synthesis was increased dose‐dependently by liposomes filled with Angiotensin II as measured by [3H]‐thymidine incorporation at high (EC50=27±6 pM) and low (EC50=14±5 nM) affinity binding sites with increases in Emax of 58±4 and 37±4% above quiescent cells, respectively. Cell growth was corroborated by an increase in cell number. Extracellular Angiotensin II (10 pM – 10 μM) did not modify [3H]‐thymidine incorporation. Growth effects of Angiotensin IIintr mediated via high affinity sites were inhibited by liposomes filled with 1 μM of the non‐peptidergic antagonists losartan (AT1‐receptor) or PD123319 (AT2‐receptor) or with the peptidergic agonist CGP42112A (AT2‐receptor). Emax values were decreased to 30±3, 29±4 and 4±2%, respectively, without changes in EC50. The Angiotensin IIintr effect via low affinity sites was only antagonized by CGP42112A (Emax=11±3%), while losartan and PD123319 increased Emax to 69±4%. Intracellular applications were ineffective in the absence of Angiotensin IIintr. Neither intracellular nor extracellular Angiotensin I (1 μM) were effective. The Angiotensin IIintr induced growth response was blocked by selective inhibition of phosphatidyl inositol 3‐kinase (PI‐3K) by wortmannin (1 μM) and of the mitogen‐activated protein kinase (MAPK/ERK) pathway by PD98059 (1 μM) to 61±14 and 4±8% of control, respectively. These data demonstrate that Angiotensin IIintr induces cell growth through atypical AT‐receptors via a PI‐3K and MAPK/ERK ‐sensitive pathway.

Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2‐purinoceptors in C2C12 myotubes

1 The presence of a nucleotide receptor and a discrete ATP‐sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP‐sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+. 2 The nucleotides ATP and UTP caused a concentration‐dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 ± 2 μm, UTP 80 ± 4 μm). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1‐, P2X‐ and P2Y‐purinoceptors, adenosine, α,β‐methylene ATP and 2‐methylthio ATP, appeared to be ineffective. 3 Under Ca2+‐free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP‐ and UTP‐induced formation of Ins(1,4,5)P3 was diminished. 4 The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP‐ and UTP‐induced Ins(1,4,5)P3 formation showed cross‐desensitization, whereas cross‐desensitization was absent in responses elicited by one of the nucleotides and bradykinin. 5 The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP‐ and UTP‐stimulated myotubes showed slopes greater than unity (1.63 ± 0.09 and 1.37 ± 0.11, respectively). Apparent pA2 values were 4.50 ± 0.48 and 4.41 ± 0.63 for ATP and UTP, respectively. 6 Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+. The ATP and UTP‐evoked increase in intracellular Ca2+ was not additive and showed cross‐desensitization. Cross‐desensitization was absent in myotubes stimulated with one of the nucleotides and bradykinin. 7 These results show that ATP‐ and UTP‐induced formation of Ins(1,4,5)P3, Ca2+ release from internal stores and Ca2+‐influx from the extracellular space are mediated exclusively via the nucleotide type P2‐purinoceptor in mouse C2C12 myotubes.

P2-purinoceptor-activated membrane currents and inositol tetrakisphosphate formation are blocked by suramin

The effect of suramin on the ATP-induced response in vas deferens DDT1 MF-2 smooth muscle cells was studied. Stimulation of P2-purinoceptors by ATP caused a change in membrane currents, measured by using the whole-cell patch-clamp configuration, and enhanced the formation of inositol phosphates, as analysed by high performance liquid chromatography. The ATP-induced membrane current consisted of a triphasic response, carried by a fast inward current, followed by a transient outward current and a sustained inward current. Inositol tetrakisphosphate (InsP4) formation increased in the presence of ATP. The formation of the isomers Ins(1,3,4,5)P4, Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 increased significantly after 5 min stimulation with ATP. Suramin inhibited the ATP-evoked membrane currents and the ATP-induced formation of inositol tetrakisphosphate isomers concentration dependently, but did not affect the basal inositol phosphate levels in the absence of ATP. These results indicate that suramin inhibits ATP-activated cellular processes in DDT1 MF-2 vas deferens cells, most likely by acting on P2-purinoceptors.

The effect of the PKC inhibitor GF109203X on the release of Ca2+ from internal stores and Ca2+ entry in DDT1 MF-2 cells

1 The effects of the specific protein kinase C (PKC) inhibitor, GF109203X, were measured on the cytoplasmic Ca2+ concentration ([Ca2+]i), and on histamine H1 receptor‐ and thapsigargin‐mediated increases in [Ca2+]i in DDT1 MF‐2 smooth muscle cells. 2 After pretreatment of cells with GF109203X (5 μm, 45 min), the histamine (100 μm)‐induced initial rise in [Ca2+]i, representing Ca2+ mobilization from internal stores, was inhibited (by 59 ± 7%). The slowly declining phase of the histamine induced Ca2+ response, reflecting Ca2+ entry, was enhanced (83 ± 26%) in the presence of the PKC inhibitor. 3 The histamine induced release of Ca2+ from internal stores, measured after blocking Ca2+ entry with LaCl3 was inhibited by GF109203X in a concentration‐dependent manner (IC50:3.1 ± 1.1 μm). 4 Histamine‐induced formation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) was not changed in the presence of GF109203X. 5 The PKC activating phorbol ester, phorbol 12‐myristate 13‐acetate (PMA, 1 μm), strongly reduced histamine‐induced Ins(1,4,5)P3 formation (58 ± 16%). This effect was reversed by GF109203X (5 μm). Furthermore, PMA diminished histamine evoked Ca2+ release (50 ± 6%) and blocked Ca2+ entry completely. 6 The rise in [Ca2+]i caused by blocking endoplasmic reticulum Ca2+‐ATPase with thapsigargin (1 μm), was strongly reduced (57 ± 3%) after pretreatment of cells with GF109203X. Downregulation of PKC by long‐term pretreatment of cells with PMA (1 μm, 48 h) did not abolish this effect of GF109203X (48 ± 3% inhibition). 7 In permeabilized DDT1 MF‐2 cells preloaded with 45Ca2+ in the presence of GF109203X, the amount of 45Ca2+ released by Ins(1,4,5)P3 (10 μm) was markedly reduced (42 ± 9%). GF109203X did not release Ca2+ itself and did not impair Ins(1,4,5)P3 receptor function. 8 Uptake of 45Ca2+ by intact cells, representing Ca2+ entry, was enhanced by GF109203X (65 ± 11%), by histamine (24 ± 6%) and also by thapsigargin (121 ± 10%). The GF109203X‐ and the thapsigargin‐induced uptake of 45Ca2+ were not additive. 9 These data suggest that GF109203X reduces the filling‐state of intracellular Ins(1,4,5)P3 sensitive Ca2+ stores by inhibiting the Ca2+ uptake into these stores, thereby promoting store‐dependent (capacitive) Ca2+ entry.

Suramin and the inhibitory junction potential in taenia caeci of the guinea-pig

The effect of surinam on the inhibitory junction potential evoked in smooth muscle cells of guinea-pig taenia caeci by stimulation of intramural nerves at 22 degrees C was investigated. The amplitude of the inhibitory junction potential was reduced concentration dependently by suramin. The suppression of this response by suramin became less pronounced when the number of stimuli increased (pulse rate: 20/s). These results indicate that suramin reduces the inhibitory junction response by interacting with P2-purinoceptors.

Characterization of P2‐purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes

1 The formation of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2C12 myotubes. 2 ATP (100 μm) and ATP7S (100 μm) caused a sustained increase in cyclic AMP content of the cells, reaching a maximum after 10 min. The cyclic AMP content reached a maximum in the presence of 100 μm ATP, followed by a decline at higher ATP concentrations. ATP‐induced cyclic AMP formation was inhibited by the P2‐purinoceptor antagonist, suramin. 3 Myotubes hydrolysed ATP to ADP at a rate of 9.7 ± 1.0 nmol mg−1 protein min−1. However, further hydrolysis of ADP to AMP and adenosine was negligible. 4 The cyclic AMP formation induced by ADP (10 μm–1 mm) showed similar characteristics to that induced by ATP, but a less pronounced decline was observed than with ATP. ADP‐induced cyclic AMP formation was blocked by suramin, while cyclic AMP formation elicited by adenosine (10 μm–1 mm) was insensitive to suramin. 5 The ATP analogue, α,β‐methylene‐ATP also induced a suramin‐sensitive cyclic AMP formation, while 2‐methylthio‐ATP and the pyrimidine, UTP, did not affect cyclic AMP levels. 6 Stimulation of the myotubes with ATP or UTP (10 μm–1 mm) caused a concentration‐dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 μm–1 mm) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels. 7 Incubation of the cells with UTP (30 μm–1 mm) inhibited the ATP‐ and ADP‐induced cyclic AMP formation, suggesting that stimulation of the ‘nucleotide’ type P2‐receptor inhibits P2‐purinoceptor mediated cyclic AMP formation in C2C12 myotubes. In contrast, UTP (30 μm–1 mm) enhanced adenosine‐induced cyclic AMP formation. 8 Adenosine‐sensitive P1‐purinoceptors activating cyclic AMP formation were found in C2C12 myotubes. Further, a novel P2‐purinoceptor is postulated, sensitive to ATP, ADP and ATPγS, which also activates the formation of cyclic AMP in C2C12 myotubes.

THE NUCLEOTIDE RECEPTORS ON MOUSE C2C12 MYOTUBES

1 The response of C2C12 mouse myotubes to stimulation with adenosine triphosphate (ATP) and other nucleotides was studied by measuring changes in membrane potential. 2 A transient hyperpolarization followed by a slowly declining depolarization of the cells was observed in the presence of ATP (10 μm−1 mm). 3 The hyperpolarization was not observed in the absence of external calcium, and was abolished in the presence of tetraethylammonium (20 mm) or the bee toxin, apamin (0.1 μm). The depolarization was reduced under low sodium conditions. 4 A biphasic change in membrane potential was also recorded in the presence of adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS) and the pyrimidine uridine triphosphate (UTP), while the ATP derivatives and analogues, adenosine diphosphate, adenosine, α,β‐methylene ATP and 2‐methylthio ATP and the nucleotides, guanosine triphosphate and cytidine triphosphate, did not affect the membrane potential of the myotubes. 5 The hyperpolarization elicited by ATPγS or UTP was also blocked by apamin and abolished under Ca2+‐free conditions. 6 In contrast to ATP and ATPγS, the depolarization evoked by UTP was unaffected under low Na+ and less sensitive to the antagonistic action of suramin. 7 The ATP and UTP responses at maximal concentration were not additive after simultaneous application. ATP elicited a depolarization if applied after UTP, while UTP did not change membrane potential following the application of ATP. 8 The concentration‐response curves of the effective nucleotides were shifted to the right in the presence of suramin, suggesting competitive antagonism. 9 These results can be explained by the presence of ‘nucleotide receptors’ mediating the ATP/UTP‐induced hyperpolarization and depolarization in C2C12 myotubes. Furthermore, an increase in Na+‐conductivity can be exclusively activated by ATP.

Suramin reverses non-depolarizing neuromuscular blockade in rat diaphragm

Unexpectedly, it was observed that the P2-purinoceptor antagonist, suramin (10 microM to 1 mM), reversed the muscle paralysis caused by structurally unrelated non-depolarizing relaxants. Suramin competitively reversed the blocking action of pancuronium. Both the pre- and postsynaptic blockade of nicotinic receptors by pancuronium was counteracted, as shown by the action of suramin, using train-of-four stimulation. Suramin did not affect the paralysis caused by the depolarizing relaxant, succinylcholine. The reversal action of suramin was not due to an increase in the acetylcholine concentration in the synaptic cleft, since neither the contraction of preparations partially paralysed by diminished acetylcholine release in the presence of low Ca2+ or high Mg2+ nor acetylcholinesterase activity were affected. Suramin did not affect the reduction in twitch tension caused by adenosine and potentiated the ATP-induced reduction in twitch, indicating that ATP-sensitive receptors are not involved in the reversal action of suramin. Consequently, these results suggest that the action of suramin is due to binding with a site on the acetylcholine receptor also occupied by non-depolarizing relaxants, but different from the site occupied by succinylcholine.

INOSITOL PHOSPHATES FORMED IN RAT AORTA AFTER ALPHA-1-ADRENOCEPTOR STIMULATION ARE INHIBITED BY FORSKOLIN

Rat aortic smooth muscle rings without endothelial cells were subjected to alpha 1-adrenoceptor stimulation. We measured the contractile state of the smooth muscle cells and the formation of inositol phosphates (InsPs) on receptor stimulation. Using different extracellular calcium-containing solutions (2.5 mM, 0.1 mM and Ca(2+)-free) enabled us to discriminate three contractile phases after noradrenaline (10(-5) M) stimulation: an initial fast contraction (15 s) and a fast and slow component of the sustained contraction, which was established 10 min after stimulation. Under normal calcium conditions in the presence of 10 mM LiCl the formation of Ins(1,4,5)P3 was increased predominantly after stimulation, while the formation of Ins(1,3,4)P3, Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and InsP5/InsP6 was also stimulated. The cAMP-inducing agent forskolin (0.5 microM) induced a relaxation of the basal tone and increased the level of the InsP4 isomers. The noradrenaline-induced contractile responses as well as the formation of InsP fractions mentioned were inhibited by forskolin. Further an increase in the formation of phosphatidylinositol bisphosphate was observed. It is concluded that in rat aorta InsPs and in particular Ins(1,4,5)P3 is involved in the different contractile phases caused by alpha 1-adrenoceptor stimulation. The relaxation induced by forskolin under these circumstances could be explained by an interaction of forskolin, most likely via the formation of cAMP, with InsPs formation at the level of phospholipase C activation.