Characterising the developmental profile of human embryonic stem cell-derived medium spiny neuron progenitors and assessing mature neuron function using a CRISPR-generated human DARPP-32WT/eGFP-AMP reporter line.

In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFß signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32WT/eGFP-AMP-LacZ). The addition of dopamine to MSNs either increased, decreased or had no effect on intracellular calcium, indicating the presence of multiple dopamine receptor subtypes. In summary, we demonstrate greater control over early fate decisions using activin-A, Wnt and SHH to direct differentiation into MSNs. We also generate a DARPP32 reporter line that enables deeper pharmacological profiling and interrogation of complex receptor interactions in human MSNs.

Comparing mouse and human pluripotent stem cell derived cardiac cells: Both systems have advantages for pharmacological and toxicological screening.

Pluripotent stem cells offer an unparalleled opportunity to investigate cardiac physiology, pharmacology, toxicology and pathophysiology. In this paper we describe the use of both mouse (Nkx2-5(eGFP/w)) and human (NKX2-5(eGFP/w)) pluripotent stem cell reporter lines, differentiated toward cardiac lineage, for live single cell high acquisition rate calcium imaging. We also assess the potential of NKX2-5(eGFP/w) cardiac lineage cells for use toxicological screening as well as establish their sensitivity to a shift between low and high oxygen environments. Differentiated mouse Nkx2-5(eGFP/w) cells demonstrated a wide range of spontaneous oscillation rates that could be reduced by ryanodine (10µM), thapsigargin (1µM) and ZD7288 (10µM). In contrast human NKX2-5(eGFP/w) cell activity was only reduced by thapsigargin (1µM). Human cell survival was sensitive to the addition of trastuzumab and doxorubicin, while the switch from a low to a high oxygen environment affected oscillation frequency. We suggest that the human NKX2-5(eGFP/w) cells are less suitable for studies of compounds affecting cardiac pacemaker activity than mouse Nkx2-5(eGFP/w) cells, but are very suitable for cardiac toxicity studies.

A novel androgen signalling pathway uses dihydrotestosterone, but not testosterone, to activate the EGF receptor signalling cascade in prostate stromal cells.

BACKGROUND AND PURPOSE: Human prostate growth and function are tightly controlled by androgens that are generally thought to exert their effects by regulating gene transcription. However, a rapid, non-genomic steroid action, often involving an elevation of intracellular calcium ([Ca(2+) ]i ), has also been described in a number of cell types. In this study we investigate whether androgens acutely regulate [Ca(2+) ]i in stromal cells derived from the human prostate. EXPERIMENTAL APPROACH: Human-cultured prostatic stromal cells (HCPSCs) were loaded with the calcium-sensitive fluorophore, fura-2-acetoxymethyl ester (FURA-2AM) (10 µM). Changes in [Ca(2+) ]i in response to the androgens, dihydrotestosterone (DHT) and testosterone, as well as EGF were measured by fluorescence microscopy. KEY RESULTS: DHT, but not testosterone (0.03-300 nM), elicited concentration-dependent elevations of [Ca(2+) ]i within 1 min of addition. These responses were blocked by the androgen receptor antagonist, flutamide (10 µM); the sarcoplasmic reticulum ATPase pump inhibitor, thapsigargin (1 µM); the inositol trisphosphate receptor inhibitor, 2-aminoethyldiphenyl borate (50 µM) and the PLC inhibitor, U-73122 (1 µM). Responses were also blocked by the L-type calcium channel blocker, nifedipine (1 µM), and by removal of extracellular calcium. A similar transient elevation of [Ca(2+) ]i was elicited by EGF (100 ng·mL(-1) ). The EGF receptor inhibitor, AG 1478 (30 nM), and the MMP inhibitor, marimastat (100 nM), blocked the DHT-induced elevation of [Ca(2+) ]i . CONCLUSIONS AND IMPLICATIONS: These studies show that DHT elicits an androgen receptor-dependent acute elevation of [Ca(2+) ]i in HCPSC, most likely by activating EGF receptor signalling.

Novel drug targets for the pharmacotherapy of benign prostatic hyperplasia (BPH).

Benign prostatic hyperplasia (BPH) is the major cause of lower urinary tract symptoms in men aged 50 or older. Symptoms are not normally life threatening, but often drastically affect the quality of life. The number of men seeking treatment for BPH is expected to grow in the next few years as a result of the ageing male population. Estimates of annual pharmaceutical sales of BPH therapies range from $US 3 to 10 billion, yet this market is dominated by two drug classes. Current drugs are only effective in treating mild to moderate symptoms, yet despite this, no emerging contenders appear to be on the horizon. This is remarkable given the increasing number of patients with severe symptoms who are required to undergo invasive and unpleasant surgery. This review provides a brief background on prostate function and the pathophysiology of BPH, followed by a brief description of BPH epidemiology, the burden it places on society, and the current surgical and pharmaceutical therapies. The recent literature on emerging contenders to current therapies and novel drug targets is then reviewed, focusing on drug targets which are able to relax prostatic smooth muscle in a similar way to the α(1) -adrenoceptor antagonists, as this appears to be the most effective mechanism of action. Other mechanisms which may be of benefit are also discussed. It is concluded that recent basic research has revealed a number of novel drug targets such as muscarinic receptor or P2X-purinoceptor antagonists, which have the potential to produce more effective and safer drug treatments.

Midbrain and forebrain patterning delivers immunocytochemically and functionally similar populations of neuropeptide Y containing GABAergic neurons.

Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca(2+) imaging and [(3)H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), L-glutamate (30 µM), noradrenaline (30 µM), acetylcholine (30 µM) and dopamine (30 µM), with similar elevations of intracellular Ca(2+)([Ca(2+)](i)). The presence of GABA(A) and GABA(B) antagonists, bicuculline (30 µM) and CGP55845 (1 µM), increased the elevation of [Ca(2+)](i) in response to dopamine (30 µM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [(3)H]-GABA release from forebrain and midbrain cultures. Dopamine (30 µM) did not stimulate significant [(3)H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA(+) neurons contain NPY, and show similar agonist-induced elevations of [Ca(2+)](i) and [(3)H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates.

Endothelin-1 and angiotensin II modulate rate and contraction amplitude in a subpopulation of mouse embryonic stem cell-derived cardiomyocyte-containing bodies.

Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have applications in understanding cardiac disease pathophysiology, pharmacology, and toxicology. Comprehensive characterization of their basic physiological and pharmacological properties is critical in determining the suitability of ESC-CMs as models of cardiac activity. In this study we use video microscopy and quantitative PCR to investigate the responses of mouse ESC-CMs to adrenoceptor, muscarinic, angiotensin II (Ang II), and endothelin-1 (ET-1) receptor activation. Isoprenaline (10 nM-10 µM) increased beating rate and contraction amplitude in all beating bodies (BBs), whereas carbachol (up to 1 µM) and the I(f) channel blocker ZD-7288 (10 µM) decreased contraction frequency. ET-1 (0.01-100 nM) reduced contraction amplitude in all BBs and increased contraction frequency in 50% of BBs; these effects were blocked by the ET(A) receptor antagonist BQ123 (250 nM). Ang II (0.01 nM-1 µM) increased both contraction amplitude (all BBs) and frequency (in 50% of BBs), effects blocked, respectively, by losartan (100 nM) and PD123,319 (200 nM). These results indicate the presence of functional ET(A) and both AT1 and AT2 receptors in murine ESC-CMs, but their expression and or activity appears to be evident only in a limited set of BBs.

P2X2, P2X4 and P2Y1 receptors elevate intracellular Ca2+ in mouse embryonic stem cell-derived GABAergic neurons.

BACKGROUND AND PURPOSE: Neurons derived from mouse embryonic stem cells (mESCs) are a valuable resource for basic pharmacological research. With the exception of cardiomyocytes, there is relatively little understanding of the pharmacology of stem cell-derived differentiated cells. In this study we investigate P2 receptor agonist effects on GABAergic neurons derived from mESCs. EXPERIMENTAL APPROACH: mESCs were differentiated into GABAergic neurons in the presence of N2B27 culture medium. At day 24 of differentiation GABAergic neuronal responsiveness to purinergic agonists was investigated using calcium imaging and [3H]-GABA release studies. KEY RESULTS: Sub-populations of GABAergic neurons responded to some or all of the adenine and uracil nucleotides ATP, ADP, UTP and UDP (all 100 microM) with elevations of intracellular Ca2+ ([Ca2+]i). The number of neurons responding to ATP was reduced by suramin (100 microM), PPADS (10 microM) and MRS2179 (10 microM), but not by NF023 (10 microM). The response to ATP was modulated by extracellular Zn2+ and pH. Neurons also responded to ATP (100 microM) with the release of [3H]-GABA, an effect completely inhibited by tetrodotoxin (100 nM). Ap4A and 2-methylthioATP both elicited significant [3H]-GABA release. Reverse transcriptase PCR showed the presence of P2X1,2,3,4,5,6 and P2X7, and P2Y1,2 and P2Y6 receptors. mESCs expressed P2X2,5 and P2X7 and P2Y1,2 and P2Y6 receptors. CONCLUSIONS AND IMPLICATIONS: GABAergic neurons derived from stem cells elevate [Ca2+]i predominantly via the activation of P2X2, P2X4 and P2Y1 receptors. This study shows that mESCs generate good models of neuronal function for in vitro pharmacological investigation.

Sex steroids modulate alpha 1-adrenoceptor-stimulated Ca2+ elevation in human cultured prostatic stromal cells.

BACKGROUND: Benign prostatic hyperplasia is an age- and androgen-dependent condition of urethral compression caused by prostatic contractility and glandular enlargement. In this study we investigate whether testosterone, dihydrotestosterone and estradiol modulate the ability of human cultured prostatic stromal cells (HCPSCs) to respond to the adrenoceptor agonists, noradrenaline (30 microM) and phenylephrine (100 microM), the protein kinase C activating phorbol ester, phorbol diacetate (PDA, 10 microM), and the L-type Ca(2+) channel activator, (-)-Bay K8644 (Bay K, 10 microM) with elevations of intracellular Ca(2+) ([Ca(2+)](i)). METHODS: Cells were loaded with the Ca(2+) sensitive fluorophore, FURA-2AM (10 microM) and changes in intracellular Ca(2+) determined before and 8-12 min after ligand addition. RESULTS: Compared to steroid-free (SF) controls, the incubation of HCPSC with testosterone (30 and 300 pM) significantly increased responses to both noradrenaline and phenylephrine. Responses to Bay K were significantly reduced between 30 nM to 300 pM but responses to PDA were not greatly affected. Compared to SF the addition of estradiol (E(2), 100 pM) did not affect responses to phenylephrine. The concomitant addition of dihydrotestosterone (DHT) and E(2) (to give ratios from 1:1 to 1,000:1) elevated the responses to noradrenaline and phenylephrine at the extreme ranges. Responses to PDA and Bay K generally increased as DHT:E(2) approached unity. CONCLUSIONS: These results indicate that sex steroids modulate the activities of HCPSCs through the regulation of both receptors and signal transduction processes.

Exploiting new systems-based strategies to elucidate plant-bacterial interactions in the rhizosphere.

The rhizosphere is the site of intense interactions between plant, bacterial, and fungal partners. In plant-bacterial interactions, signal molecules exuded by the plant affect both primary initiation and subsequent behavior of the bacteria in complex beneficial associations such as biocontrol. However, despite this general acceptance that plant-root exudates have an effect on the resident bacterial populations, very little is still known about the influence of these signals on bacterial gene expression and the roles of genes found to have altered expression in plant-microbial interactions. Analysis of the rhizospheric communities incorporating both established techniques, and recently developed "omic technologies" can now facilitate investigations into the molecular basis underpinning the establishment of beneficial plant-microbial interactomes in the rhizosphere. The understanding of these signaling processes, and the functions they regulate, is fundamental to understanding the basis of beneficial microbial-plant interactions, to overcoming existing limitations, and to designing improved strategies for the development of novel Pseudomonas biocontrol strains.

Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation.

Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post-plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na(+) and tetraethylammonium (TEA)-sensitive K(+) channels. Neurons reached their maximal cell capacitance after D6-7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca(2+)-free, 2.5 mm Mg(2+) saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca(2+) levels within ES cell-derived neurons increased in response to glutamate receptor agonists l-glutamate, AMPA, N-methyl-d-aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl(-)-selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons.

A1 and A2A adenosine receptor modulation of alpha 1-adrenoceptor-mediated contractility in human cultured prostatic stromal cells.

1. This study investigated the possibility that adenosine receptors modulate the alpha(1)-adrenoceptor-mediated contractility of human cultured prostatic stromal cells (HCPSC). 2. The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamido-adenosine (NECA; 10 nm-10 microm), and the A(1) adenosine receptor selective agonist, cyclopentyladenosine (CPA; 10 nm-10 microm), elicited significant contractions in HCPSC, with maximum contractile responses of 18+/-3% and 17+/-2% reduction in initial cell length, respectively. 3. In the presence of a threshold concentration of phenylephrine (PE) (100 nm), CPA (1 nm-10 microm) caused contractions, with an EC(50) of 124+/-12 nm and maximum contractile response of 37+/-4%. The A(1) adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 100 nm) blocked this effect. In the presence of DPCPX (100 nm), NECA (1 nm-10 microm) inhibited contractions elicited by a submaximal concentration of PE (10 microm), with an IC(50) of 48+/-2 nm. The A(2A) adenosine receptor-selective antagonist 4-(2-[7-amino-2-[furyl][1,2,4]triazolo[2,3-alpha][1,3,5,]triazin-5-yl amino]ethyl)phenol (Zm241385 100 nm) blocked this effect. 4. In BCECF-AM (10 microm)-loaded cells, both CPA (100 pM-1 microm) and NECA (100 pm-10 microm) elicited concentration-dependent decreases in intracellular pH (pH(i)), with EC(50) values of 3.1+/-0.3 and 6.0+/-0.3 nm, respectively. The response to NECA was blocked by Zm241385 (100 nm; apparent pK(B) of 9.4+/-0.4), but not by DPCPX (100 nm). The maximum response to CPA was blocked by DPCPX (100 nm), and unaffected by Zm241385 (100 nm). 5. NECA (10 nm-10 microm) alone did not increase [(3)H]-cAMP in HCPSC. In the presence of DPCPX (100 nm), NECA (10 nm-10 microm) caused a concentration dependent increase in [(3)H]-cAMP, with an EC(50) of 1.2+/-0.1 microm. This response was inhibited by Zm241385 (100 nm). CPA (10 nm-10 microm) had no effect on cAMP, in the presence or absence of forskolin (1 microm). 6. These findings are consistent with a role for adenosine receptors in the modulation of adrenoceptor-mediated contractility in human prostate-derived cells.

Alpha 1-adrenoceptor effects mediated by protein kinase C alpha in human cultured prostatic stromal cells.

1 We have investigated the effects of alpha(1)-adrenoceptor stimulation upon contractility, Ca(2+) influx, inositol phosphate production, and protein kinase C (PKC) translocation in human cultured prostatic stromal cells (HCPSC). 2 The alpha(1)-adrenoceptor selective agonist phenylephrine elicited contractile responses of HCPSC, i.e. a maximal cell shortening of 45+/-6% of initial cell length, with an EC(50) of 1.6+/-0.1 microM. The alpha(1)-adrenoceptor selective antagonists prazosin (1 microM) and terazosin (1 microM) both blocked contractions to phenylephrine (10 microM). The L-type calcium channel blocker nifedipine (10 microM), and the PKC inhibitors Gö 6976 (1 microM) and bisindolylmaleimide (1 microM) also inhibited phenylephrine-induced contractions. 3 Phenylephrine caused a concentration dependent increase in inositol phosphate production (EC(50) 119+/-67 nM). This response was blocked by terazosin (1 microM). 4 Phenylephrine caused the translocation of the PKC alpha isoform, but not the beta, delta, gamma, epsilon or lambda isoforms, from the cytosolic to the particulate fraction of HCPSC, with an EC(50) of 5.7+/-0.5 microM. 5 In FURA-2AM (5 microM) loaded cells, phenylephrine elicited concentration dependent increases in [Ca(2+)](i), with an EC(50) of 3.9+/-0.4 microM. The response to phenylephrine (10 microM) was blocked by prazosin (1 microM), bisindolymaleimide (1 microM), and nifedipine (10 microM). 6 In conclusion, this study has shown that HCPSC express functional alpha(1)-adrenoceptors, and that the intracellular pathways responsible for contractility may be largely dependent upon protein kinase C activation and subsequent opening of L-type calcium channels.

Testosterone- and phorbol ester-stimulated proliferation in human cultured prostatic stromal cells.

Prostatic stromal proliferation may be commonly associated with the development of benign prostatic hyperplasia. In this study, we investigate the role of testosterone and protein kinase C in stimulating cultured stromal cell proliferation. Testosterone increased the uptake of [(3)H]-thymidine into the human cultured prostatic stromal cells, this was reduced by the protein kinase C inhibitors, bisindolylymaleimide (10 nM) and myristoylated protein kinase C inhibitor (mPKCi, 20 microM), but not by Gö 6983 (1 microM) or Gö 6976 (1 microM). Cells responded to the addition of the PKC activators phorbol 12,13 dibutyrate (PDB), phorbol 12,13 diacetate (PDA), 12-deoxyphorbol 13-acetate (DPA) and 12-deoxyphorbol 13-tetradecanoate (DPT) with proliferation (order of potency DPT> or =PDB>>PDA=DPA). The DPT-stimulated proliferative response was inhibited after cells were electroporated with PKCalpha antisense, but not mismatch oligonucleotides (8 microM). These results indicate that PKCalpha is involved in the proliferative response of human cultured prostatic stromal cells.

A(2A) adenosine receptor mediated potassium channel activation in rat epididymal smooth muscle.

The effects of A(2) adenosine receptor agonists upon phenylephrine-stimulated contractility in preparations of rat epididymis were investigated. Preparations responded to phenylephrine (3 microM) with submaximal contractions. Adenosine and the stable agonists 5'-N-ethylcarboxamido-adenosine (NECA) and 2-p-(2-carboxyethyl) phenethylamino-N-ethylcarboxamide adenosine (CGS 21680) inhibited phenylephrine-induced contractions (potency order, NECA>CGS 21680>adenosine). The A(2A) receptor-selective antagonist, 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo-[2,3-a][1,3, 5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 30 microM) blocked the response to NECA. The A(2A) adenosine receptor-mediated inhibitory responses to NECA were reduced by the K(ATP) channel blocker, glibenclamide (3 microM) and abolished by charybdotoxin (100 nM). The diterpene forskolin elicited a concentration-dependent inhibition of phenylephrine (3 microM)-stimulated contractility (by 62+/-8% of control at 100 microM). Charybdotoxin (100 nM), but not glibenclamide (3 microM) blocked the forskolin (10 microM) inhibition of phenylephrine-stimulated contractility. NECA elicited concentration-dependent increases in both cyclic AMP and cyclic GMP accumulation which were antagonized by ZM 241385 (30 nM). The protein kinase G activator, APT-cyclic GMP (8-(-Aminophenylthio) guanosine-3',5'-cyclic monophosphate) and the protein kinase A activator (Sp)-8-bromoadenosine-3',5'-cyclic monophosphorothioate (Sp-8-Br-cyclic AMPs), inhibited phenylephrine (3 microM) induced contractions of rat epididymis. Glibenclamide (3 microM), but not charybdotoxin (100 nM), inhibited ATP-cyclic GMP responses. Charybdotoxin (100 nM), but not glibenclamide (3 microM) reduced the effect of Sp-8-Br-cyclic AMPs. This study shows that the A(2A) adenosine receptor inhibition of epididymal contractility may be mediated through the activation of charybdotoxin- and glibenclamide-sensitive potassium channels and may involve the activation of both protein kinases A and G.

Gi-Protein alpha-subunit mRNA antisense oligonucleotide inhibition of Gi-coupled receptor contractile activity in the epididymis of the guinea-pig.

We have used a reversible permeabilization method to facilitate the entry of Gialpha1, 2 and 3 G-protein subunit mRNA antisense or mismatch oligonucleotides into intact tissue, to investigate the G-protein alpha-subunit coupling of alpha2-adrenoceptors, neuropeptide Y (NPY) Y1, and A1 adenosine receptors in preparations of the epididymis of the guinea-pig. The alpha2-adrenoceptor agonist, xylazine, elicited concentration dependent contractions from preparations of phenylephrine (3 microM)-stimulated epididymis (pEC50 value 6.52+/-0.39, maximum response 236+/-41 mg force). Compared to respective mismatch controls the incubation of preparations with Gialpha2, but not with Gialpha1 or Gialpha3 mRNA antisense oligonucleotides (30 microM) reduced the maximal xylazine-potentiation of phenylephrine (3 microM)-stimulated contractility (to 51+/-12% of Gialpha2 mismatch control). The oligonucleotide incubations had no effect upon the pEC50 values of xylazine. The A1 adenosine receptor agonist, cyclopentyladenosine (CPA) elicited concentration dependent contractions from preparations of phenylephrine (3 microM)-stimulated epididymis (pEC50 value 7.66+/-0.57, maximum response 208+/-54 mg force). Incubation of preparations of epididymis with Gialpha1, but neither Gialpha2 nor Gialpha3 antisense oligonucleotides reduced the maximal CPA-potentiation of phenylephrine (3 microM)-stimulated contractions (to 55+/-17% of Gialpha1 mismatch control), pEC50 values were not affected. The incubation of preparations with Gialpha2 antisense mRNA oligonucleotides reduced the maximal NPY-potentiation of phenylephrine (3 microM)-stimulated contractions (to 62+/-15% of Gialpha mismatch control). Compared with Gialpha2 mismatch controls, the incubation of preparations with Gialpha1 and Gialpha3 oligonucleotides also reduced the NPY-potentiation of phenylephrine (3 microM)-stimulated contractions. These studies indicate that, in the guinea-pig epididymis, alpha2-adrenoceptors and A1 adenosine receptors preferentially couple to effectors through Gialpha2 and Gialpha1 subunits respectively. In contrast NPY receptors may elicit effects through either Gialpha1, 2 or 3 subunits.

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig.

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [3H]-cyclic adenosine monophosphate ([3H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The alpha1-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca2+ channel blocker, nifedipine (10 microM) the non-selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA, 1 microM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml(-1) 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7+/-0.9 fold). 2. In the presence of phenylephrine (1 microM), NECA and the A1 adenosine receptor selective agonists, N6-cyclopentyladenosine (CPA) and (2S)-N6-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC50 values 8.18+/-0.19, 7.79+/-0.29 and 8.15+/-0.43 respectively). The A3 adenosine receptor agonists N6-iodobenzyl-5'-N-methylcarboxamido adenosine (IBMECA) and N6-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 microM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pKB 8.75+/-0.88) and the maximal response to NECA was reduced. 3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 microM) stimulated contractions (pIC50 7.15+/-0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 microM) and the A2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-++ +ylamino]ethyl)phenol (ZM 241385; 30 nM). 4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 microM)-stimulated accumulation of [3H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17+6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [3H]-cyclic AMP in preparations of epididymis (pEC50 values 5.35+/-0.35 and 6.42+/-0.40 respectively), the NECA-induced elevation of [3H]-cyclic AMP was antagonised by XAC (apparent pKB 6.88+/-0.88) and also by the A2A adenosine receptor antagonist, ZM 241385 (apparent pKB 8.60+/-0.76). 5. These studies are consistent with the action of stable adenosine analogues at post-junctional A1 and A2 adenosine receptors in the epididymis of the guinea-pig. A1 Adenosine receptors potentiate alpha1-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A2 adenosine receptors, possibly of the A2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.