Catalin M. Filipeanu

Angiotensin-Converting Enzyme 2 Overexpression in the Subfornical Organ Prevents the Angiotensin II–Mediated Pressor and Drinking Responses and Is Associated With Angiotensin II Type 1 Receptor Downregulation

We recently reported the presence of angiotensin-converting enzyme (ACE)2 in brain regions controlling cardiovascular function; however, the role of ACE2 in blood pressure regulation remains unclear because of the lack of specific tools to investigate its function. We hypothesized that ACE2 could play a pivotal role in the central regulation of cardiovascular function by regulating other renin–angiotensin system components. To test this hypothesis, we generated an adenovirus expressing the human ACE2 cDNA upstream of an enhanced green fluorescent protein (eGFP) reporter gene (Ad-hACE2-eGFP). In vitro characterization shows that neuronal cells infected with Ad-hACE2-eGFP (10 to 100 multiplicities of infection), but not Ad-eGFP (100 multiplicities of infection), exhibit dose-dependent ACE2 expression and activity. In addition, an active secreted form was detected in the conditioned medium. In vivo, Ad-hACE2-eGFP infection (2×106 plaque-forming units intracerebroventricularly) produced time-dependent expression and activity (with a peak at 7 days) in the mouse subfornical organ. More importantly, 7 days after virus infection, the pressor response to angiotensin (Ang) II (200 pmol intracerebroventricularly) was significantly reduced in Ad-hACE2-eGFP–treated mice compared with controls. Furthermore, subfornical organ–targeted ACE2 overexpression dramatically reduced the Ang II–mediated drinking response. Interestingly, ACE2 overexpression was associated with downregulation of the Ang II type 1 receptor expression both in vitro and in vivo. These data suggest that ACE2 overexpression in the subfornical organ impairs Ang II–mediated pressor and drinking responses at least by inhibiting the Ang II type 1 receptor expression. Taken together, our results show that ACE2 plays a pivotal role in the central regulation of blood pressure and volume homeostasis, offering a new target for the treatment of hypertension and other cardiovascular diseases.

DIFFERENTIAL EXPRESSION OF SPHINGOLIPIDS IN MRP1 OVEREXPRESSING HT29 CELLS

We have obtained a novel multidrug resistant cell line, derived from HT29 G+ human colon carcinoma cells, by selection with gradually increasing concentrations of the anti‐mitotic, microtubule‐disrupting agent colchicine. This HT29col cell line displayed a 25‐fold increase in colchicine resistance and exhibited cross‐resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT‐PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29col cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5‐carboxyfluorescein, an increased efflux activity of HT29col cells was measured, as compared to the wild‐type HT29 G+ cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5‐carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29col cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P‐glycoprotein‐specific substrate. Colchicine‐induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis‐related signal transduction processes, was much more abundant in HT29col cells, which is indicative of a stress response. Int. J. Cancer 87:172–178, 2000.

Multiple effects of tyrosine kinase inhibitors on vascular smooth muscle contraction

The effects of three tyrosine kinase inhibitors: genistein, quercetin and psi-tectorigenin, were investigated on contractions evoked in de-endothelised rat aortic rings, either by phenylephrine or 70 mM K+. A dose-dependent inhibition of both contractions by all three compounds was observed, the phenylephrine-mediated contractions being more sensitive to genistein. No differences between genistein or quercetin effects in pre-treatment or post-treatment protocols were found. Ca2+ store refilling, expressed in terms of phenylephrine-induced tension in Ca(2+)-free medium, was dose-dependently blocked by quercetin and genistein. Sodium orthovanadate, an inhibitor of tyrosine phosphatase, contracted the rat aortic rings with an IC50 of 0.66 microM. Its presence during the refilling period after exposure to Ca(2+)-free medium completely prevented the subsequent response to phenylephrine. One can conclude that the use of the above-mentioned protein tyrosine kinase inhibitors in the rat aorta blocks a step involved in Ca2+ entry and Ca2+ store refilling. A definite conclusion regarding the vanadate effects is not possible due to the fact that this compound also affects Ca2+ ATP-ases.

Enhancement of the Recycling and Activation of β-Adrenergic Receptor by Rab4 GTPase in Cardiac Myocytes

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous β-adrenergic receptor (β-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. β1-AR, the predominant subtype of β-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized β-AR to the cell surface and enhanced β-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of β-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the β-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous β-AR and augmentation of Rab4-mediated traffic enhances β-AR function in cardiac myocytes.

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.

Differential regulation of the cell-surface targeting and function of β- and α1-adrenergic receptors by Rab1 GTPase in cardiac myocytes

The molecular mechanism underlying the export from the endoplasmic reticulum (ER) to the cell surface and its role in the regulation of signaling of adrenergic receptors (ARs) remain largely unknown. In this report, we determined the role of Rab1, a Ras-like GTPase that coordinates protein transport specifically from the ER to the Golgi, in the cell surface targeting and function of endogenous β- and α1-ARs in neonatal rat ventricular myocytes. Adenovirus-driven expression of Rab1 into myocytes selectively increased the cell-surface number of α1-AR, but not β-AR, whereas the dominant-negative mutant Rab1N124I significantly reduced the cell-surface expression of β-AR and α1-AR. Brefeldin A inhibited β-AR and α1-AR export and antagonized the Rab1 effect on α1-AR expression. Manipulation of Rab1 function similarly influenced the transport of α1A- and α1B-ARs as well as β1- and β2-ARs. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA induced a marked accumulation of GFP-tagged β2-AR and α1B-AR in the ER. Consistent with the effects on receptor cell-surface targeting, Rab1 selectively enhanced ERK1/2 activation and hypertrophic growth in response to the α1-AR agonist phenylephrine but not to the β-AR agonist isoproterenol. Rab1N124I inhibited both agonist-mediated ERK1/2 activation and hypertrophic growth in neonatal myocytes. These results demonstrate that the cell-surface targeting and signaling of β- and α1-ARs require Rab1 and are differentially modulated by augmentation of Rab1 function. Our data provide strong evidence implicating the ER-to-Golgi traffic as a site for selective manipulation of distinct AR function in cardiac myocytes.

Urotensin-II regulates intracellular calcium in dissociated rat spinal cord neurons

Urotensin‐II (U‐II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U‐II on [Ca2+]i was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase‐positive and had morphological features of motoneurons. U‐II induced [Ca2+]i increases in these neurons with a threshold of 10−9 m, and a maximal effect at 10−6 m with an estimated EC50 of 6.2 × 10−9 m. The [Ca2+]i increase induced by U‐II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+‐free medium. Omega‐conotoxin GVIA (10−7 m), a N‐type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega‐conotoxin GVIIC (10−7 m) and the l‐type Ca2+ channel blocker, verapamil (10−5 m) had minimal effects. Down‐regulation of protein kinase C by 4‐α‐phorbol 12‐myristate 13‐acetate (10−6 m) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10−6 m) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10−6 m) or Rp‐8‐pCPT‐cGMPS (3 × 10−5 m) did not modify U‐II‐induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10−6 m) and Rp‐cAMPS (3 × 10−5 m) reduced the response to 25 ± 3% and 42 ± 8%, respectively. Present results demonstrate that U‐II modulates [Ca2+]i in rat spinal cord neurons via protein kinase A cascade.

Δ9-Tetrahydrocannabinol activates [Ca2+]i increases partly sensitive to capacitative store refilling

Delta9-tetrahydrocannabinol induces [Ca2+]i increases in DDT1MF-2 smooth muscle cells. Both Ca2+ entry and release from intracellular Ca2+ stores were concentration dependently activated. The Ca2+ entry component contributed most to the increases in [Ca2+]i. Stimulation with delta9-tetrahydrocannabinol after functional downregulation of intracellular Ca2+ stores by longterm thapsigargin treatment, still induced a major Ca2+ entry and a minor Ca2+ release component. Thapsigargin sensitive influx and release were selectively inhibited by the cannabinoid CB1 receptor antagonist SR141716A. No effects on [Ca2+]i were obtained after stimulation with the CB2 receptor agonist palmitoylethanolamide. This study is the first demonstration of (1) Ca2+ release from thapsigargin sensitive intracellular stores and capacitative Ca2+ entry via CB1 receptor stimulation and of (2) an additional delta9-tetrahydrocannabinol induced thapsigargin insensitive component, mainly representing Ca2+ influx which is neither mediated by CB1 nor CB2 receptor stimulation.

Endoplasmic reticulum export of adrenergic and angiotensin II receptors is differentially regulated by Sar1 GTPase

The molecular mechanism underlying the export of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) remains largely unknown. In this manuscript, we investigated the role of Sar1 GTPase, which coordinates the assembly and budding of COPII-coated vesicles, in the cell-surface targeting, signaling and ER export of alpha(2B)-adrenergic (alpha(2B)-AR), beta(2)-AR and angiotensin II type 1 receptors (AT1R). The cell-surface expression of alpha(2B)-AR, beta(2)-AR and AT1R, and receptor-mediated ERK1/2 activation were significantly attenuated by the GTP-bound mutant Sar1H79G, suggesting that export from the ER of these receptors is mediated through the Sar1-dependent COPII-coated vesicles. Interestingly, subcellular distribution analyses showed that alpha(2B)-AR and AT1R were highly concentrated at discrete locations near the nucleus in cells expressing Sar1H79G, whereas beta(2)-AR exhibited an ER distribution. These data indicate that Sar1-catalyzed efficient GTP hydrolysis differentially regulates ER export of adrenergic and angiotensin II receptors. These data provide the first evidence indicating distinct mechanisms for the recruitment of different GPCRs into the COPII vesicles on the ER membrane.

Long-term behavioral and pharmacodynamic effects of delta-9- tetrahydrocannabinol in female rats depend on ovarian hormone status

Abuse of Δ9‐THC by females during adolescence may produce long‐term deficits in complex behavioral processes such as learning, and these deficits may be affected by the presence of ovarian hormones. To assess this possibility, 40 injections of saline or 5.6 mg/kg of Δ9‐THC were administered i.p. daily during adolescence to gonadally intact or ovariectomized (OVX) female rats, yielding four treatment groups (intact/saline, intact/THC, OVX/saline, and OVX/THC). Δ9‐THC (0.56–10 mg/kg) was then re‐administered to each of the four groups during adulthood to examine their sensitivity to its disruptive effects. The behavioral task required adult subjects to both learn (acquisition component) different response sequences and repeat a known response sequence (performance component) daily. During baseline (no injection) and control (saline injection) sessions, OVX subjects had significantly higher response rates and lower percentages of error in both behavioral components than the intact groups irrespective of saline or Δ9‐THC administration during adolescence; the intact group that received Δ9‐THC had the lowest response rates in each component. Upon re‐administration of Δ9‐THC, the groups that received adolescent ovariectomy alone, adolescent Δ9‐THC administration alone, or both treatments were found to be less sensitive to the rate‐decreasing effects, and more sensitive to the error‐increasing effects of Δ9‐THC than the control group (i.e. intact subjects that received saline during adolescence). Neurochemical analyses of the brains from each adolescent‐treated group indicated that there were also persistent effects on cannabinoid type‐1 (CB‐1) receptor levels in the hippocampus and striatum that depended on the brain region and the presence of ovarian hormones. In addition, autoradiographic analyses of the brains from adolescent‐treated, but behaviorally naïve, subjects indicated that ovariectomy and Δ9‐THC administration produced effects on receptor coupling in some of the same brain regions. In summary, chronic administration of Δ9‐THC during adolescence in female rats produced long‐term effects on operant learning and performance tasks and on the cannabinoid system that were mediated by the presence of ovarian hormones, and that altered their sensitivity to Δ9‐THC as adults.