Margaret A. Scofield

Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis

Excessive activation of G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) pathways has been linked to prostate cancer metastasis. Rac activation by guanine nucleotide exchange factors (GEFs) plays an important role in directional cell migration, a critical step of tumor metastasis cascades. We found that the upregulation of P-Rex1, a Rac-selective GEF synergistically activated by Gβγ freed during GPCR signaling, and PIP3, generated during either RTK or GPCR signaling, strongly correlates with metastatic phenotypes in both prostate cancer cell lines and human prostate cancer specimens. Silencing endogenous P-Rex1 in metastatic prostate cancer PC-3 cells selectively inhibited Rac activity and reduced cell migration and invasion in response to ligands of both epidermal growth factor receptor and G-protein-coupled CXC chemokine receptor 4. Conversely, expression of recombinant P-Rex1, but not its ‘GEF-dead’ mutant, in non-metastatic prostate cancer cells, such as CWR22Rv1, increased cell migration and invasion through Rac-dependent lamellipodia formation. More importantly, using a mouse xenograft model, we showed that the expression of P-Rex1, but not its mutant, induced lymph node metastasis of CWR22Rv1 cells without an effect on primary tumor growth. Thus, by functioning as a coincidence detector of chemotactic signals from both GPCRs and RTKs, P-Rex1-dependent activation of Rac promotes prostate cancer metastasis.

Protein kinase C mediates P2U purinergic receptor inhibition of K+ channel in apical membrane of strial marginal cells

Strial marginal cells (SMC) electrogenically secrete K+ via slowly activating K+ (I[sK]) channels, consisting of I(sK) regulatory and KvLQT1 channel subunits, and the associated short circuit current (I[sc]) is inhibited by agonists of the apical P2U receptor [Liu et al., Audit. Neurosci. 2 (1995) 331-340]. Measurements of relative K+ flux (JK) with a self-referencing K+-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macro patch recordings from the apical membrane of gerbil SMC showed a decrease of the I(sK) channel current (I[IsK]) by 88 +/- 8% during pipette perfusion of 100 microM ATP. The magnitude of the decrease of L(sc) by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (20 nM) decreased I(IsK) (gerbil: by 62 +/- 10%; rat: by 72 +/- 6%) in perforated-patch whole-cell recordings while the inactive analog, 4alphaPMA, had no effect. By contrast, elevation of cytosolic [Ca2+] by A23187 increased the whole-cell I(IsK). The expression of the isk gene transcript was confirmed and the serine responsible for the species-specific response to PKC was found to be present in the gerbil I(sK) sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the I(sK) channel of SMC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human cochlea may be controlled by PKC in the same way as in our animal model.

Evidence for a calcium-sensing receptor in the vascular smooth muscle cells of the spiral modiolar artery.

Abstract. The vascular diameter of the gerbilline spiral modiolar artery has been shown to depend on the presence of extracellular Ca2+ but it remained unknown whether the smooth muscle cells of this arteriole contain a Ca2+ sensing receptor (CaSR). The cytosolic Ca2+ concentration ([Ca2+]i) was monitored as fluo 3 fluorescence and the vascular diameter was measured by video-microscopy in isolated in vitro superfused spiral modiolar arteries. RT-PCR was used to probe for the presence of CaSR transcripts. Increasing the extracellular Ca2+ concentration ([Ca2+]o) from 1 to 10 mm caused a biphasic increase in [Ca2+]i that was paralleled by a vasoconstriction. The initial rate of this vasoconstriction, 2.01 ± 0.07 μm/sec (n= 131), was inhibited when cytosolic Ca2+ stores were presumably depleted with thapsigargin (IC50= 3 × 10−9m, n= 26) or ryanodine (IC50= 4 × 10−8m, n= 25) or when PLC was inhibited by 10−6m U73122 (n= 8). The initial rate of this constriction was not affected by the L-type Ca2+ channel blocker 10−6m nifedipine (n= 5), by 10−6m U73343 (n= 6), which is the inactive analogue of U73122, by the T-type Ca2+ channel blocker 10−6 Gd3+ (n= 6) or the Na+/Ca2+ exchanger blocker 10−4m Ni2+ (n= 5). The agonist rank potency order was Gd3+ > Ni2+ > Ca2+ >> neomycin = Mg2+. Analysis of RNA isolated from the SMA revealed a RT-PCR product of the appropriate size for the CaSR (448 bp). Sequence analysis of the amplified cDNA fragment revealed a 94–96% amino acid identity compared to other CaSRs. These results demonstrate that the spiral modiolar artery contains a CaSR, which is most likely located in the vascular smooth muscle cells.

P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells.

Vestibular dark cells (VDC) are known to electrogenically secrete K+ via slowly activating K+ (IsK) channels, consisting of IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current (Isc) is inhibited by agonists of the apical P2U (P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1995). Measurements of relative K+ flux (JK) with a self-referencing K(+)-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macropatch recordings from gerbil VDC showed a decrease of the IsK channel current (IIsK) by 83 +/- 7% during pipette perfusion of 10 microM ATP. The magnitude of the decrease of Isc by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased IIsK by 79 +/- 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4 alpha-PMA, had no effect. In contrast, elevation of cytosolic Ca2+ concentration by A-23187 increased the whole cell IIsK. The expression of the isk gene transcript was confirmed, and the serine responsible for the species-specific response to PKC was found to be present in the gerbil IsK sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the IsK channel of VDC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human vestibular system may be controlled by PKC in the same way as in our animal model.Vestibular dark cells (VDC) are known to electrogenically secrete K+ via slowly activating K+(IsK) channels, consisting of IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current ( I sc) is inhibited by agonists of the apical P2U(P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1995). Measurements of relative K+ flux ( J K) with a self-referencing K+-selective probe demonstrated a decrease in J K after apical perfusion of 100 μM ATP. On-cell macropatch recordings from gerbil VDC showed a decrease of the IsKchannel current ( I IsK) by 83 ± 7% during pipette perfusion of 10 μM ATP. The magnitude of the decrease of I scby ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased I IsK by 79 ± 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4α-PMA, had no effect. In contrast, elevation of cytosolic Ca2+ concentration by A-23187 increased the whole cell I IsK . The expression of the isk gene transcript was confirmed, and the serine responsible for the species-specific response to PKC was found to be present in the gerbil IsKsequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the IsK channel of VDC in response to activation of the apical P2Ureceptor and predict that the secretion of endolymph in the human vestibular system may be controlled by PKC in the same way as in our animal model.

K+ secretion in strial marginal cells is stimulated via beta 1-adrenergic receptors but not via beta 2-adrenergic or vasopressin receptors.

Abstract. Pharmacologic tools were used to identify receptors in functional studies by measuring either transepithelial current (Isc) in strial marginal cells (SMC) or cAMP production in stria vascularis (SV). Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed using tissues isolated from gerbils unless specified otherwise. Isc under control conditions was 1090 ± 21 μA/cm2 (n= 213) in gerbil SMC and 2001 ± 95 μA/cm2 (n= 6) in murine SMC. Direct stimulation of adenylate cyclase with 10-5m forskolin but not with 10−5m 1,9-dideoxy-forskolin resulted in an increase in the Isc by a factor of 1.14 ± 0.01 (n= 6). The vasopressin-receptor agonist 10−8m Arg8-vasopressin had no significant effect on Isc in gerbil and murine SMC. The β-adrenergic agonists isoproterenol, norepinephrine and epinephrine stimulated Isc with an EC50 of (6 ± 2) × 10−7m (n= 28), (3 ± 1) × 10−6m (n= 40) and (7 ± 2) × 10−6m (n= 38), respectively. Isoproterenol stimulated cAMP production in SV with an EC50 of (5 ± 2) × 10−7m (n= 8). The β-antagonist 10−4m propanolol completely inhibited 2 × 10−5m isoproterenol-induced stimulation of Isc. The β-antagonists atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of Isc with a KDB of 1 × 10−7m (pKDB= 6.96 ± 0.15, n= 14), 1 × 10−7m (pKDB= 7.01 ± 0.14, n= 15), 2 × 10−9m (pKDB= 8.73 ± 0.13, n = 19), respectively. CGP20712A inhibited isoproterenol-induced cAMP production with a KDB of 1 × 10−10m (pKDB= 9.94 ± 0.55, n= 9). RT-PCR of total RNA isolated from SV using primers specific for the β1-, β2- and β3-adrenergic receptors revealed products of the predicted sizes for the β1- and β2- but not the β3-adrenergic receptor. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that K+ secretion in SMC is under the control of β1-adrenergic receptors but not β2-adrenergic or vasopressin-receptors and that the β1-subtype is the primary β-adrenergic receptor in SV although SV contains transcripts for both β1- and β2-adrenergic receptors.

α1A-Adrenergic receptors mediate vasoconstriction of the isolated spiral modiolar artery in vitro

Several lines of evidence suggest that cochlear blood flow is under the control of the sympathetic nervous system and that this control is mediated via alpha-adrenergic receptors. The goal of the present study was to determine whether alpha-adrenergic receptors mediate vasoconstriction of the spiral modiolar artery and, if so, to determine which subtype dominates this response. Vascular diameter was measured with video microscopy in the isolated superfused spiral modiolar artery in vitro. The diameter of the spiral modiolar artery under control conditions was 61 +/- 2 microm (n = 60). Spontaneous vasomotion was observed in most specimens. Addition of norepinephrine to the superfusate caused a phasic vasoconstriction and an increase in the amplitude of vasomotion. These effects were limited to the vicinity of arteriolar branch points of the spiral modiolar artery. Norepinephrine-induced vasoconstriction occurred with EC50 of (1.9 +/- 0.4) x 10(-5) M (n = 44) and the vascular diameter was maximally reduced by a factor of 0.87 +/- 0.01 (n = 29). Neither the phasic nature nor the EC50 of the norepinephrine-induced vasoconstrictions was altered in the presence of the beta2-adrenergic receptor antagonist 10(-5) M ICI118551 or the nitric oxide synthase inhibitor 10(-4) M NOARG. In contrast, the alpha2-adrenergic receptor antagonist 10(-7) M yohimbine and the alpha1-adrenergic receptor antagonist 10(-9) and 10(-8) M prazosin caused a significant shift in the dose-response curve. The affinity constants (K(DB)) for yohimbine and prazosin were (5+/-2) x 10(-8) M (n=4) and (2.0+/-0.7) x 10(-10) M (n=18), respectively. The alpha1A-adrenergic receptor antagonist 10(-8) M 5-methyl urapidil and the alpha1D-adrenergic receptors antagonist 5 x 10(-6) M BMY7378 caused a significant shift in the dose-response curve. The K(DB) values for 5-methyl urapidil and for BMY7378 were (2.7 +/- 0.7) x 10(-10) M (n = 8) and (4.4 +/- 2.7) x 10(-7) M (n = 8), respectively. Further, total RNA was isolated from microdissected spiral modiolar arteries and the presence of transcripts for alpha1-adrenergic receptor subtypes was determined by reverse transcription polymerase chain reaction (RT-PCR). Primers specific for gerbil alpha1-adrenergic receptor subtypes were developed using RNA from rat and gerbil brain. Analysis of RNA extracted from the spiral modiolar artery revealed RT-PCR products of the appropriate size for the alpha1A-adrenergic receptor, however, no evidence for the alpha1B- and alpha1D-adrenergic receptor was found. Further, analysis of RNA extracted from blood, which was a contaminant of the microdissected spiral modiolar arteries, revealed no RT-PCR products. Sequence analysis of the RT-PCR product of the alpha1A-adrenergic receptor from the spiral modiolar artery confirmed its identity. Identity between the 175 nt gerbil sequence fragment and the known rat, mouse and human alpha1A-adrenergic receptor sequences was 90.9, 92.0 and 85.2%, respectively. These observations demonstrate that the spiral modiolar artery contains alpha1A-adrenergic receptors which mediate vasoconstriction at branch points.

β1-Adrenergic Receptors but not β2-Adrenergic or Vasopressin Receptors Regulate K+ Secretion in Vestibular Dark Cells of the Inner Ear

Abstract. Receptors were identified pharmacologically in functional studies where K+ secretion was monitored as transepithelial current (Isc). Further, receptors were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Isc under control conditions was 796 ± 15 μA/cm2 (n= 329) in gerbilline VDC and 900 ± 75 μA/cm2 (n= 6) in murine VDC. Forskolin (10−5m) but not 1,9-dideoxy-forskolin increased Isc by a factor of 1.42 ± 0.05 (n= 7). 10−9m Arg8-vasopressin and 10−9m desmopressin had no significant effect in gerbilline and murine VDC. Isoproterenol, norepinephrine, epinephrine and prenalterol stimulated Isc maximally by a factor of 1.38 ± 0.04 (n= 7), 1.59 ± 0.06 (n= 6), 1.64 ± 0.03 (n= 8) and 1.37 ± 0.03 (n= 6), respectively. The EC50 values were (1.4 ± 0.7) × 10−8m (n= 36), (2.5 ± 1.0) × 10−8m (n= 31), (1.7 ± 0.7) × 10−7m (n= 36) and (5 ± 4) × 10−7m (n= 32), respectively. Propanolol inhibited isoproterenol-induced stimulation of Isc. Atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of Isc with a pKDB of 5.0 × 10−8m (pKDB= 7.30 ± 0.07, n= 38), 4.4 × 10−8m (pKDB= 7.36 ± 0.14, n= 37) and 6.8 × 10−12m (pKDB= 11.17 ± 0.12, n= 37), respectively. RT-PCR of total RNA isolated from microdissected vestibular labyrinth tissue using specific primers revealed products of the predicted sizes for β1- and β2-adrenergic receptors but not for β3-adrenergic receptors. Sequence analysis of the amplified cDNA fragments from gerbilline tissues revealed a 96.4%, 91.5% and 89.6% identity compared to rat β1-, β2- and β3-adrenergic receptors, respectively. These results demonstrate that K+ secretion in VDC is under the control of β1- but not β2- or β3-adrenergic receptors or vasopressin-receptors.

CGRP receptors in the gerbil spiral modiolar artery mediate a sustained vasodilation via a transient cAMP-mediated Ca2+-decrease

Alteration of cochlear blood flow may be involved in the etiology of inner ear disorders like sudden hearing loss, fluctuating hearing loss and tinnitus. The aim of the present study was to localize the vasodilator calcitonin gene-related peptide (CGRP) and to identify CGRP receptors and their signaling pathways in the gerbil spiral modiolar artery (SMA) that provides the main blood supply of the cochlea. CGRP was localized in perivascular nerves by immunocytochemistry. The vascular diameter and cytosolic Ca2+ concentration [Ca2+]i in the smooth muscle cells were measured simultaneously with videomicroscopy and fluo-4-microfluorometry. Calcitonin receptor-like receptor (CRLR) mRNA was identified by RT-PCR as a specific 288 bp fragment in total RNA isolated from the vascular wall. The SMA was preconstricted by a 2-min application of 1 nM endothelin-1 (ET1). CGRP, forskolin, and dibutyryl-cAMP caused a vasodilation (EC50 = 0.1 nM, 0.3 mM, and 20 mM). CGRP and forskolin caused an increase in cAMP production and a transient decrease in the [Ca2+]i. The CGRP-induced vasodilation was antagonized by CGRP8-37 (KDB = 2 mM). The K+-channel blockers iberiotoxin and glibenclamide partially prevented the CGRP- or forskolin-induced vasodilations but failed to reverse these vasodilations. These results demonstrate that CGRP is present in perivascular nerves and causes a vasodilation of the ET1-preconstricted SMA. The data suggest that this vasodilation is mediated by an increase in the cytosolic cAMP concentration, a transient activation of iberiotoxin-sensitive BK and glibenclamide-sensitive KATP K+ channels, a transient decrease in the [Ca2+]i and a long-lasting Ca2+ desensitization.

Distribution of alpha1-adrenergic receptor mRNA species in rat heart.

Radioligand binding studies have detected alpha1A- and alpha1B-adrenergic receptors (AR) in rat heart, but the ligands available for these studies lack the sensitivity and specificity needed to map possible differences in alpha1-AR subtype expression. We therefore used competitive reverse transcriptase-polymerase chain reaction (RT-PCR) techniques to measure steady-state amounts of alpha1-AR messenger RNA (mRNA) subtypes in tissue dissected from several regions of rat heart. We detected mRNA for alpha1A-, alpha1B-, and alpha1D-AR in each region. Irrespective of the alphaAR subtype, the total number of alpha1-AR transcripts has the following regional rank order: left ventricular papillary muscle > left ventricle > left atrium > apex > right ventricle > ventricular septum > right atria. Among the regions, the fractional contribution of alpha1A-, alpha1B-, and alpha1D-AR mRNA to the total amount of alpha1-AR displays considerable variability. The alpha1B-AR mRNA accounts for >50% of the total alpha1-AR mRNA in all regions except the ventricular septum. There are also significant percentages of alpha1A-AR in each region, especially in the papillary muscle (48%) and ventricular septum (48%). The alpha1D-AR mRNA transcripts are found in comparatively low numbers; their highest levels (18% of total) were found in the right ventricle. These differences in alpha1-AR mRNA expression may contribute to the observed regional differences in myocardial responses to alpha1-AR agonists and antagonists.

Identification of mRNA and protein expression of the Na/K-ATPase α1-, α2- and α3-subunit isoforms in Antarctic and New Zealand nototheniid fishes

Abstract Antarctic nototheniids living in waters below 0 °C have a serum osmolality nearly double that of temperate nototheniids. Warm acclimation of Antarctic nototheniids to +4 °C decreases serum osmolality by 25% and nearly doubles gill Na/K-ATPase activity without an increase in Na/K-ATPase number or a change in the enzymes affinity for ouabain. However, the increased activity may result from a change in gill Na/K-ATPase α-subunit isoform composition. Four known Na/K-ATPase α-subunit isoforms have been identified in other species, however, identification of the isoforms in teleosts has been incomplete. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and cloning, we have identified partial cDNA sequences of the Na/K-ATPase α1-, α2- and α3-subunit isoforms in the Antarctic nototheniid Trematomus bernacchii . Using isoform-specific primers, the mRNA for all three isoforms was found in T. bernacchii brain, gill, heart, trunk kidney and muscle. Using isoform-specific antibodies, the Na/K-ATPase α1-, α2- and α3-subunit isoforms were found in the tissues of both T. bernacchii and Notothenia angustata , a temperate nototheniid. This is the first report detecting all three isoforms at the nucleotide and protein level in teleosts. Further experiments will determine if the gill T. bernacchii Na/K-ATPase α-subunit isoform composition changes during warm acclimation.