Involvement of signal transducing GTP-binding proteins in renal artery alpha 1-adrenoceptor mediated smooth muscle contraction.

OBJECTIVE: To determine the participation of GTP-binding proteins (G-proteins) in the cellular mechanism of the phenylephrine-induced renal artery vasospasm by using swine renal artery smooth muscle rings in a standard organ baths, as increased noradrenaline release from perivasal and intramural sympathetic nerve endings during renal ischaemia results in increased vascular smooth muscle tone that is important in the loss of kidney function during renal transplantation and nephron-sparing surgery. MATERIALS AND METHODS: Fresh swine kidneys were transported in cold calcium-free Tyrode solution to the laboratory. Adipose tissue around the arteries was removed, the organ de-capsulated and interlobar arteries dissected. The contractile properties of renal artery smooth muscle rings were assessed in a standard organ bath, the rings pre-tensioned at 2 g. Contractions were evoked by applying the alpha 1-adrenoceptor selective agonist phenylephrine (1 nmol/L to 0.3 mmol/L). Isometric contractions of the tissue were registered and stored digitally. Dose-response curves were obtained sequentially with a wash-out of 20 min between each concentration; the maximum contractility of an individual muscle ring was set at 100%. Dose-response curves of inhibitory agents (e.g. WB4101, cholera and pertussis toxins) were determined by comparing the remaining contractility after incubating with the respective drug with a control contraction that was evoked three times (10 mumol/L phenylephrine) and the mean set at 100%. RESULTS: Phenylephrine induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 100 nmol/L and an EC50 of 0.8 mumol/L. The receptor was identified as the alpha 1A-subtype by the selective antagonist WB4101. Pre-treatment of tissue rings with 5 micrograms/mL pertussis toxin (120 min, 37 degrees C) inhibited the control contraction by a mean (SEM) of 52.0 (4.6)%, whereas pre-treatment with 1 microgram/mL cholera toxin (60 min, 37 degrees C), leading to a permanent activation of the Gs-protein via blockade of the GTPase activity, decreased the response by 39.0 (8.2%). CONCLUSION: These results suggest a coupling of alpha 1A-adrenoceptors in renal vascular tissue to the heterotrimeric Gs-protein and to heterotrimeric G-proteins of the G1- and/or G0-family in the phenylephrine-induced contraction.

Involvement of cyclic nucleotides in renal artery smooth muscle relaxation.

The elevation of vascular smooth muscle tone in the renal arteries during kidney transplantation and nephron-sparing surgery plays a major role in postsurgical organ dysfunction. Therefore, a better understanding of the intracellular mechanisms of contraction and relaxation is of fundamental interest to improve urological treatment. The present study was designed to investigate the complex intracellular system of cyclic nucleotides involved in the regulation of smooth muscle relaxation by using swine renal artery rings in the Schuler organ bath. Phenylephrine (PE) induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 10 nM and an EC(50) of 804 nM. The receptor was identified as alpha(1A)-subtype by the selective antagonist WB4101. Increasing the intracellular concentration of cyclic 3':5'-adenosine monophosphate (cAMP) by dibutyryl-cAMP (5 mM) and forskolin (5 micro M) resulted in a decreased contractiltity of 48.0% and 76.3%, respectively. Elevation of the cytosolic content of cyclic 3':5'-guanosine monophosphate (cGMP) using dibutyryl-cGMP (1 mM), sodium nitroprusside (100 micro M) and SIN-1 (100 micro M) decreased the average PE-induced contraction by 16.4%, 41.9% and 62.4%, respectively. The unselective phosphodiesterase inhibitors theophylline (1 mM), papaverine (100 micro M) and IBMX (5 mM) reduced the PE-induced contraction by 37.3%, 93.1% and 95.5%, respectively. Furthermore, selective inhibition of phosphodiesterases by milrinone (PDE(3)-selective) resulted in a decreased contractility by 1.3% (50 micro M), 29.5% (100 micro M) and 93.5% (5 mM), and using rolipram (PDE(4) selective), the PE-induced contraction was inhibited by 57.9% (50 micro M) and 81.9% (100 micro M). The results suggest the involvement of cAMP and cGMP in the relaxation of renal artery smooth muscle cells. Moreover, phosphodiesterases, especially PDE(3) and PDE(4), seem to play a critical role in the regulation of renal artery smooth muscle tone.

Regulation of renal artery smooth muscle tone by alpha1-adrenoceptors: role of voltage-gated calcium channels and intracellular calcium stores.

The ischemia induced vasospasm of the renal arterial blood vessels mediated by alpha1-adrenoceptors is of importance for the loss of kidney function. This is based on reduced perfusion of the kidney cortex occurring in kidney transplant and organ preserving surgery. The present study considered the intracellular mechanism of the norepinephrine (NE) induced renal artery vasospasm by using swine renal artery smooth muscle ring. Norepinephrine and phenylephrine (PE) induced dose-dependent and fully reversible isometric contractions with a threshold concentration of 10 nM (n = 7) and 10 nM (n = 4), and an EC50 of 0.3 microM and 1 microM, respectively. The receptor was identified as alpha1A-subtype. The contraction was completely inhibited by verapamil (IC50 = 1.51 microM; n = 11) and diltiazem (IC50 = 9.49 microM; n = 8) and 85% by nifedipine (IC50 = 0.13 microM; n = 21). Blockade of the intracellular inositol- 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store by thapsigargin (1 microM, n = 7) or suppression of Ca2+ release from the intracellular Ca2+-sensitive Ca2+ store by ryanodine (100 microM, n = 4) inhibited the PE induced contraction by 39.5% and 47.6%, respectively. The results suggest a key role of voltage-dependent Ca2+ channels and intracellular Ca2+ stores in the alpha1A-adrenoceptor induced contraction of the renal artery.

Regulation of prostatic smooth muscle contractility by intracellular second messengers: implications for the conservative treatment of benign prostatic hyperplasia.

The increased sympathetic neurotransmission in benign prostatic hyperplasia (BPH) results in a alpha 1C-adrenoceptor-mediated increase in prostatic smooth muscle tone which seems to be responsible for the dynamic infravesical obstruction occurring in BPH. The prostatic smooth muscle contractions evoked by norepinephrine can be efficiently blocked by alpha 1-adrenoceptor blockers. Moreover, an impressive number of clinical trials illustrated the beneficial results of alpha 1-adrenoceptor blockers in the treatment of BPH. However, despite knowledge of alpha 1-adrenergic neurotransmission and the clinical application of its blockade by selective alpha 1-adrenoceptor antagonists, very little is known about the intracellular pathways involved in the regulation of prostatic smooth muscle contractility. To study the intracellular mechanism of the alpha 1C-adrenoceptor-induced prostatic smooth muscle contraction, the patch-clamp technique in the whole-cell configuration mode combined with the Fura-II fluorescence technique was used in human, enzymatically isolated smooth muscle cells obtained from patients undergoing transurethral resection of the prostate because of symptomatic BPH. Furthermore changes in prostatic smooth muscle contractility were registered in organ bath experiments. Application of the selective alpha 1-adrenoceptor agonist phenylephrine (PE) increased the L-type Ca(2+)-channel current (ICa) dose dependently from 8 up to 18.5 microA/cm2, simultaneously elevating the free cytoplasmic Ca2+ concentration ([Ca2+]i) up to 1.9 microM. Pretreating the myocytes with pertussis toxin, an exotoxin of Bordetella pertussis which inactivates GTP-binding proteins (G proteins) of the Gi and G(o) family by ADP ribosylation, reduced the PE-induced ICa stimulation by 71.5 +/- 5.6% (n = 21). Dialysis of the cytosol with the second messenger inositol-1,4,5-trisphosphate (IP3), which releases Ca2+ from intracellular non-mitochondrial, IP3-sensitive Ca2+ pools, imitated the PE-evoked responses. Pretreating the myocytes with the Ca(2+)-release blockers ryanodine (10-100 microM, n = 8), thapsigargin (0.1 microM, n = 11) or low-molecular weight heparin (n = 14) largely attenuated the PE-evoked responses. The experimental results suggest a coupling of alpha 1-adrenoceptors to phospholipase C-converting phosphoinositol-4,5-bisphosphate into diacylglycerol, an endogenous activator of the protein kinase C and IP3 which releases Ca2+ from intracellular stores stimulating ICa via Ca(2+)-calmodulin-dependent protein kinase induced phosphorylation of voltage-dependent Ca2+ channels. This knowledge could be of interest for conservative treatment in symptomatic BPH.

Smooth muscle contractility in prostatic hyperplasia: role of cyclic adenosine monophosphate.

The role of cyclic 3'-5' adenosine monophosphate (cAMP) on alpha 1-adrenoceptor (alpha 1-receptor) induced smooth muscle contractions in symptomatic benign prostatic hyperplasia (BPH) was investigated. Application of the selective alpha 1-receptor agonist phenylephrine (PE) induced fully reversible contractions in a dose-dependent fashion. Phosphodiesterase (PDE) inhibitors blocking the degradation of cAMP suppressed the PE induced contractions as follows: theophylline (1 mM), 91.1 +/- 1.4%; papaverine (0.5 mM), 822.8 +/- 3.2%; milrinone (0.5 mM), 68.2 +/- 0.6%. Forskolin (50 microM), which elevates cAMP through direct activation of adenylatecyclase (AC), inhibited the PE induced contractions by 82.4 +/- 3.6%. To further increase the intracellular cAMP concentration ([cAMP]i), the membrane permeable cAMP analogue N6-2'-O-dibutyryladenosine derivative (dBcAMP; 1 mM) was applied and reduced the PE evoked contractions by 69.8 +/- 2.3%. We conclude that elevation of [cAMP]i is an important step in inducing smooth muscle relaxation.

G-proteins in alpha 1-adrenoceptor mediated prostatic smooth muscle contraction.

The role of signal transducing guanine-nucleotide binding proteins (G-proteins) in alpha 1-receptor mediated smooth muscle contractions was investigated in human hyperplastic prostatic tissue. The selective alpha 1-receptor agonist phenylephrine (PE) evoked dose dependent contractions antagonized by the alpha 1-receptor blockers prazosin (EC50 10 nM) and YM617 (EC50 3 nM). Application of nifedipine (1-10,000 nM), a blocker of voltage-dependent L-type Ca(2+)-channels (VDCC), inhibited the PE evoked contraction up to 65.4%. Pretreating the tissue strips with pertussis toxin (PTX, exotoxin from Bordetella pertussis; 5-25 micrograms/ml), inactivating a subpopulation of G-proteins, inhibited the PE induced contractions up to 73.9%. PTX pretreatment had no effect on contractions elicited by 125 mM K+. Application of nifedipine to PTX pretreated tissue led to an additional inhibition of 13.7%. Our findings demonstrate the involvement of PTX-sensitive G-proteins in the signal transduction pathway of alpha 1-receptor induced contractions of prostatic smooth muscle. The remaining contractility of PTX pretreated tissue suggests additional participation of PTX insensitive mechanisms in alpha 1-receptor mediated prostatic smooth muscle contractions.

Alpha-1 receptor mediated smooth muscle regulation in benign prostatic hyperplasia.

Symptoms in benign prostatic hyperplasia (BPH) are partially caused by an increased prostatic smooth muscle tone. This tone depends on extra- and intracellular Ca2+ stores and is regulated by various second messenger pathways. We investigated the role of intracellular Ca2+ stores and cyclic 3'-5' adenosine monophosphate (cAMP) in BPH. Contractions elicited by the specific alpha 1-receptor agonist phenylephrine (PE) were inhibited by the selective alpha 1-receptor antagonists prazosin and YM 617. To elucidate the contribution of intracellular Ca2+ stores to the alpha 1-receptor induced contraction nifedipine, a blocker of voltage dependent L-type Ca2+ channels (VDCC) was applied and found to inhibit the PE induced contractions up to 65%. To further confirm the participation of intracellular Ca2+ stores, we applied ryanodine (10 microM) which reduced the alpha 1-receptor mediated contractions up to 80%. The remaining contraction was sensitive to nifedipine. The cAMP pathway mediating smooth muscle relaxation by regulating intracellular Ca2+ concentrations ([Ca2+]i) was also investigated. Nonspecific phosphodiesterase (PDE) inhibitors such as papaverine (0.5 mM) and theophylline (1 mM) and the specific PDE inhibitor milrinone (0.5 mM), all of which prevent degradation of cAMP, suppressed the PE induced contractions by 82%, 91% and 68%, respectively. Forskolin (50 microM), an activator of adenylylecyclase (AC), inhibited the PE induced contractions by 83%. The membrane permeable cAMP analog, N6-2'-0-dibutyryladenosine derivative (dBcAMP) also reduced the PE induced response by 70%.

Role of intracellular Ca2+ stores in smooth muscle contractions of the guinea pig vas deferens.

Guinea pig vas deferens was used as an animal model for alpha-1 adrenoceptor (alpha 1-receptor) mediated contractions in human hyperplastic prostatic tissue. The selective alpha 1-receptor agonist, phenylephrine (PE), induced fully reversible, dose-dependent contractions antagonized by increasing concentrations of the alpha 1-receptor blockers prazosin (1-100 nM) and YM 617 (0.1-10 nM). Removal of extracellular Ca2+ reduced PE-evoked contractions in a time-dependent manner. Nifedipine (1-1000 nM), a blocker of voltage-dependent L-type Ca2+ channels (VDCC), inhibited the PE-induced response by up to 65%. Removal of extracellular Ca2+ abolished the alpha 1-agonist reactivity in a time-dependent fashion. To elucidate the participation of intracellular Ca2+ stores in alpha 1-receptor-mediated contractions, the tissue was pretreated with ryanodine (10 microM) or thapsigargin (0.1 microM), established inhibitors of Ca2+ release from intracellular pools. Both substances reduced the PE contractions by up to 80%. Nifedipine suppressed the remaining contractions completely. This provides evidence that Ca2+ influx through VDCC and Ca2+ release from intracellular stores contribute to alpha 1-receptor-mediated contractions in the guinea pig vas deferens and may be important in obstructive benign prostatic hyperplasia.