Biomedical subjects

John M Haynes

Publications and source records attributed to John M Haynes.

9 recordsLinked to original sources

PubMed2006

Phosphorylation of the PKG substrate, vasodilator-stimulated phosphoprotein (VASP), in human cultured prostatic stromal cells.

Nitric oxide (NO) is known to regulate contractility and proliferation of cells within the prostate, however, the mechanism by which this occurs is unknown. The cGMP-dependent protein kinase (PKG) signalling pathway may be involved, and recent work has shown that activation of this pathway can be assessed by analysis of phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The aim of the current study is to characterise the expression of VASP in the human prostate and human cultured prostatic stromal cells (HCPSCs), and to investigate whether NO activates PKG in these cells. Our studies revealed that VASP is expressed, and that incubation of HCPSCs with PKG-activating cGMP-analogues or the NO-donor, SNP, caused a significant PKG-dependent increase in VASP serine-239 phosphorylation. In addition, SNP elicited a reduction in intracellular K(+) in a time frame consistent with the phosphorylation of VASP and activation of PKG. These data demonstrate that VASP can be used to assess the NO/cGMP/PKG signalling pathway in HCPSCs. In addition, we demonstrate for the first time that SNP, probably via NO release, leads to phosphorylation of VASP in a manner consistent with PKG activation.

Aged↗

PubMed2006

Protein kinase G-induced activation of K(ATP) channels reduces contractility of human prostate tissue.

BACKGROUND: Human cultured prostatic stromal cells respond to protein kinase G (PKG) activators and the nitric oxide donor, sodium nitroprusside (SNP) by opening ATP-sensitive potassium channels (K(ATP) channels) to reduce nifedipine-sensitive phorbol ester-induced contractility. METHODS: PKG activators, SNP, diazoxide, nifedipine, isoprenaline, forskolin, and Sp-8-Br-cAMP were used to inhibit alpha(1)-adrenoceptor-induced contractions in tissue from transurethral resections of the prostate (TURP). The selective K(ATP) and large conductance Ca(2+) activated K(+) (BK(Ca)) channel inhibitors, glibenclamide and charybdotoxin, respectively were used to inhibit responses to PKG activators. RT-PCR identified the K(ATP) channel subunits present in TURP tissue and cultured cells. RESULTS: The PKG activators, APT-cGMP (1 nM-100 microM) and PET-cGMP (1 nM-100 microM), and also SNP (1 nM-100 microM), forskolin (10 microM), diazoxide (100 microM) and nifedipine (3 microM) inhibited phenylephrine (20 microM)-induced contractions. The effect of APT-cGMP (1 nM-100 microM) could be reversed by glibenclamide, but not by charybdotoxin. TURP tissue contained mRNA for PKG Ialpha, Ibeta, and II and the K(ATP) channel subunits Kir6.1, Kir6.2, SUR2B, and SUR1. Cultured stromal cells contained only Kir6.1 and SUR2B subunit mRNA. SUR1 mRNA was detected in one of five cultured epithelial cell lines. CONCLUSIONS: PKG activators reduce alpha(1)-adrenoceptor-induced contractility in TURP tissue via the activation of K(ATP) channels. (c) 2005 Wiley-Liss, Inc.

Aged↗

PubMed2006

Saw palmetto is an indirectly acting sympathomimetic in the rat-isolated prostate gland.

BACKGROUND: To investigate whether saw palmetto that inhibits alpha1-adrenoceptor binding in vitro affects contractility of the rat prostate gland. METHODS: The effects of a commercially available saw palmetto extract were examined on the contractility of rat-isolated prostate glands. The extract was tested in the presence and absence of phentolamine, prazosin, yohimbine, propranolol, hexamethonium, cocaine, desipramine, nifedipine, guanethidine, atropine, and alpha,beta-methylene ATP to evaluate the mechanism of action. Isolated preparations of rat vas deferens and bladder were used for comparison. RESULTS: Unexpectedly, saw palmetto extract caused contractions of the rat prostate gland that could be attenuated by prazosin, phentolamine, nifedipine, guanethidine, cocaine, and desipramine but not by any of the other pharmacological tools. Similar contractile effects were observed in rat-isolated vas deferens preparations but not in rat-isolated bladder preparations. CONCLUSIONS: In the rat prostate gland, saw palmetto extract causes indirect alpha1-adrenoceptor-mediated contractions via the release of noradrenaline from sympathetic neurons.

Amphetamine↗

PubMed2005

Pharmaceutical applications of embryonic stem cells.

Embryonic stem cells (ESCs) will become a source of all adult differentiated cells once reliable protocols for directed differentiation have been established. This resource will revolutionise laboratory cell biology and will provide much improved cell culture models for discovery and development of drugs, and fundamental studies of the genetic basis of disease. These are early days, and there are few examples of practical outcomes in the pharmaceutical world, though in recent years some drug-like molecules, which direct differentiation, have been discovered. At this stage the focus is on understanding the signalling systems and transcription factors that drive differentiation, on characterisation and isolation of precursor cells, and on establishing methods to improve the homogeneity of differentiated cells derived from ESCs. There is particular interest in establishing methods for deriving fully differentiated human cells from human ESCs (HESCs), but there are challenges to be met before HESC technology can be taken up in a widespread manner. Protocols for expansion of HESC cultures are labour-intensive at present. There is an urgent need for discovery of factors that will allow serum-free (and feeder cell-free) expansion of HESCs, and which can be applied to the majority of HESC lines. Our expectation is that adequate protocols will emerge in the coming years opening the way for development of many cell culture tools in the pharmaceutical industry. Here we review the current status and future prospects for this exciting field and encourage pharmaceutical scientists to play a role in directing its development.

Animals↗

PubMed2005

Current models of human prostate contractility.

1. The human prostate is a compact gland contributing to seminal fluid. With increasing age, most humans will develop benign prostatic hyperplasia, a condition of prostatic enlargement and contractility that leads to occlusion of the urethra. Over many years, investigators have used a variety of animal and cell culture models to elucidate some of the contractile and proliferative mechanisms that may be associated with the development of this condition. 2. This review briefly assesses the current state of knowledge of the mechanisms underlying human prostatic contractility and compares it with that of animal and cell culture models. It is not intended as a comprehensive methodological review, nor is it intended to indicate our preferences for either model. Our aim is to correlate findings from animal and cell culture models with the current understanding of human prostate contractility. 3. We hope that the present review will increase awareness of the suitability of the current models in developing our understanding of benign prostatic hyperplasia.

Animals↗

PubMed2004

Protein kinase G II-mediated proliferative effects in human cultured prostatic stromal cells.

This study investigates the effect of protein kinase G (PKG) activation upon proliferation of human cultured prostatic stromal cells. The PKG II activator (8-pCPT-cGMP; IC50 of 113+/-42 nM) and the phosphodiesterase inhibitor, zaprinast (up to 50 microM), but not the PKG I isoform activators (APT-cGMP and PET-cGMP), reduced foetal calf serum-stimulated proliferation. The effect of 8-pCPT-cGMP (30 microM) was blocked by Rp-8-Br-cGMPS (5 microM) and Rp-8-pCPT-cGMP (5 microM), but not Rp-cAMPS (5 microM). 8-pCPT-cGMP (30 microM) and zaprinast (50 microM), but not PET-cGMP (30 microM), caused a significant increase in atypical nuclei and an increase in annexin-V staining. These data indicate that activation of PKG II induces apoptosis of human cultured prostatic stromal cells.

Aged↗

PubMed2002

Phorbol ester-induced contractility and Ca2+ influx in human cultured prostatic stromal cells.

In this study, we investigated the effects of protein kinase C (PKC)-activating phorbol esters upon Ca(2+) influx and contractility in human cultured prostatic stromal cells. Tissue obtained from patients undergoing transurethral resection of the prostate was used to generate explant cultures of prostatic stromal cells. These cells expressed detectable levels of PKCalpha, delta, gamma, lambda, and zeta, but not epsilon, iota, mu, or theta; isoforms and responded to both phorbol 12,13-diacetate (PDA) and 12-deoxyphorbol 13-tetradecanoate (DPT) with concentration-dependent contractions (pEC50+/-SEM 7.07+/-0.41 and 6.39+/-0.27, respectively). The L-type Ca2+ channel blocker nifedipine (3 microM), and the PKC inhibitors Gö 6976, Gö 6983 (both 100 nM), myristoylated PKC inhibitor 19-27 (20 microM) and bisindolylmaleimide (1 microM) all abolished PDA-stimulated (1 microM) contractions. Neither PDA nor DPT (at 1 microM) caused translocation of any PKC isoform from the cytosolic to the particulate fraction. Nifedipine (3 microM), myristoylated PKC inhibitor 19-27 (20 microM), and bisindolylmaleimide (1 microM) inhibited PDA-stimulated Ca2+ influx into FURA-2 loaded cells. This study indicates that human cultured prostatic stromal cells respond to phorbol esters with contractions that are dependent upon the influx of Ca2+ through L-type Ca2+ channels and that this effect may be independent of the translocation of PKC from cytosolic to particulate fractions.

Aged↗

PubMed2002

Protein kinase G activation of K(ATP) channels in human-cultured prostatic stromal cells.

In this study, we identify and investigate the role of protein kinase G (PKG) in cells cultured from human prostatic stroma. Cells were used for immunocytochemistry, contractility or K(+) fluorescent imaging studies. All cultured prostatic stromal cells showed PKG immunostaining. Phorbol 12,13 diacetate (PDA, 1 microM) elicited contractions from human-cultured prostatic stromal cells that could be blocked by both the L-type Ca(2+) channel blocker, nifedipine (3 microM), and the protein kinase C inhibitor, bisindolylmaleimide (1 microM). The nitric oxide donor, sodium nitroprusside (SNP, molar pIC(50) 5.16+/-0.17) and the cGMP-phosphodiesterase inhibitor, zaprinast (50 microM), inhibited PDA (1 microM)-induced contractions. The PKG activator beta-phenyl-1, N(2)-ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP, molar pIC(50) 6.96 +/- 0.25) also inhibited PDA (1 microM)-induced contractions. Glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not iberiotoxin (100 nM) or Rp-cAMP (5 microM), reversed this inhibition. In human-cultured prostatic stromal cells loaded with the K(+) fluorescent indicator, 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis [(acetyloxy) methyl] ester (PBFI), PET-cGMP (300 nM) caused a reduction in intracellular K(+) that was blocked by glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not by iberiotoxin (100 nM). These data are consistent with the hypothesis that, in human-cultured prostatic stromal cells, PKG inhibits contractility through the activation of K(ATP) channels.

Adenosine Triphosphate↗

PubMedDate not supplied

Purinoceptor-mediated contractility of the perfused uterine vasculature of the guinea-pig: influence of oestradiol and pregnancy.

1. The effects of ATP, the stable ATP analogues alpha,beta-methylene ATP (alpha,beta-mATP), 2-methylthioATP (2meSATP) and adenosine tetraphosphate (ATP4), the pyrimidine nucleotide uridine 5'-triphosphate (UTP) and the alpha1-adrenoceptor agonist phenylephrine were examined on the isolated perfused uterine vasculature of dioestrous, oestradiol-treated, dexamethasone-treated and late-pregnant guinea-pigs. 2. The alpha1-adrenoceptor agonist phenylephrine elicited concentration-dependent vasoconstriction from preparations of perfused uterine vasculature from dioestrous, estradiol-treated and late-pregnant guinea-pigs. The mean maximal response to phenylephrine was unaffected by treatment of dioestrus guinea-pigs with oestradiol or dexamethasone, but was reduced in preparations from late-pregnant animals. 3. In perfused uterine arteries from dioestrous animals, the pyrimidine UTP, but not ATP4 and ATP, elicited vasoconstrictor responses. In preparations from oestradiol-treated animals, all three agonists elicited vasoconstriction, with a rank order of potency of ATP4 = UTP >> ATP, whereas in preparations from late-pregnant animals this order of potency was ATP4 >> UTP = ATP. In preparations from dexamethasone-treated animals, the vasoconstriction was similar to that seen in dioestrous animals. Vasoconstrictor responses to ATP4 were significantly greater in preparations of uterine vasculature from oestradiol-treated and pregnant animals than in preparations from dioestrous animals or dexamethasone-treated animals. 4. In preparations from dioestrous, oestradiol-treated, pregnant and dexamethasone-treated animals, alpha,beta-mATP was approximately two to three orders of magnitude more potent than 2meSATP. Compared with preparations from dioestrous animals, the maximal responses to alpha,beta-mATP were significantly greater in tissues from oestradiol-treated and pregnant animals. In preparations from dioestrous animals, the P2 purinoceptor antagonist suramin (100 micro mol/L) inhibited the responses to alpha,beta-mATP, but not those to ATP4. 5. The present study has demonstrated that pregnancy, but not the steroid treatment of dioestrous guinea-pigs with oestradiol or dexamethasone, reduces the sensitivity of the guinea-pig isolated perfused uterine vasculature to phenylephrine. In contrast, preparations from pregnant or oestradiol-treated guinea-pigs respond to ATP4 and to alpha,beta-mATP with significantly greater constrictions than those of dioestrous or dexamethasone-treated animals. These data indicate that the sensitivity of the uterine vasculature to adrenoceptor and purinoceptor agonists is differentially regulated by oestradiol and pregnancy, but not by the synthetic glucocorticoid dexamethasone.

Animals↗