Modeling the response of a biofilm to silver-based antimicrobial.

Biofilms are found within the lungs of patients with chronic pulmonary infections, in particular patients with cystic fibrosis, and are the major cause of morbidity and mortality for these patients. The work presented here is part of a large interdisciplinary effort to develop an effective drug delivery system and treatment strategy to kill biofilms growing in the lung. The treatment strategy exploits silver-based antimicrobials, in particular, silver carbene complexes (SCC). This manuscript presents a mathematical model describing the growth of a biofilm and predicts the response of a biofilm to several basic treatment strategies. The continuum model is composed of a set of reaction-diffusion equations for the transport of soluble components (nutrient and antimicrobial), coupled to a set of reaction-advection equations for the particulate components (living, inert, and persister bacteria, extracellular polymeric substance, and void). We explore the efficacy of delivering SCC both in an aqueous solution and in biodegradable polymer nanoparticles. Minimum bactericidal concentration (MBC) levels of antimicrobial in both free and nanoparticle-encapsulated forms are estimated. Antimicrobial treatment demonstrates a biphasic killing phenomenon, where the active bacterial population is killed quickly followed by a slower killing rate, which indicates the presence of a persister population. Finally, our results suggest that a biofilm with a ready supply of nutrient throughout its depth has fewer persister bacteria and hence may be easier to treat than one with less nutrient.

Nanoparticle deposition onto biofilms.

We develop a mathematical model of nanoparticles depositing onto and penetrating into a biofilm grown in a parallel-plate flow cell. We carry out deposition experiments in a flow cell to support the modeling. The modeling and the experiments are motivated by the potential use of polymer nanoparticles as part of a treatment strategy for killing biofilms infecting the deep passages in the lungs. In the experiments and model, a fluid carrying polymer nanoparticles is injected into a parallel-plate flow cell in which a biofilm has grown over the bottom plate. The model consists of a system of transport equations describing the deposition and diffusion of nanoparticles. Standard asymptotic techniques that exploit the aspect ratio of the flow cell are applied to reduce the model to two coupled partial differential equations. We perform numerical simulations using the reduced model. We compare the experimental observations with the simulation results to estimate the nanoparticle sticking coefficient and the diffusion coefficient of the nanoparticles in the biofilm. The distributions of nanoparticles through the thickness of the biofilm are consistent with diffusive transport, and uniform distributions through the thickness are achieved in about four hours. Nanoparticle deposition does not appear to be strongly influenced by the flow rate in the cell for the low flow rates considered.

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

Angiotensin-(1-7) regulates the levels of angiotensin II receptor subtype AT1 mRNA differentially in a strain-specific fashion.

Ang-(1-7) is an effector peptide of the renin-angiotensin system with several distinct actions that are likely mediated by a specific receptor. Regulatory effects of angiotensin (Ang) peptides, Ang-(1-7) and Ang II, on Ang receptor subtype 1 (AT1) mRNA expression were investigated in vascular smooth muscle cells (VSMC) from four University of Akron (Akr) rat strains (WKY, SHR and two backcross consomic lines SHR/y and SHR/a), and in SHR and WKY cells from Charles River Laboratories (Crl). In WKY/Akr and SHR/Akr, Ang-(1-7) treatment increased the levels of AT1 mRNA. This effect was inhibited by the specific Ang-(1-7) antagonist, A-779, in WKY/Akr but not SHR/Akr. Ang II had no effect in Akr cells, but it down-regulated AT1 mRNA in WKY/Crl and SHR/Crl VSMC. Ang-(1-7) did not affect AT1 mRNA levels in Crl lines. In conclusion, Ang-(1-7) regulates the AT1 receptor either directly or indirectly in a strain-specific fashion. The Ang-(1-7) antagonist, A-779, blocks the actions of Ang-(1-7) only in VSMC from WKY/Akr rats, suggesting either that the binding sites for Ang-(1-7) have different properties in SHR/Akr and WKY/Akr cell lines, or that some of the effects of Ang-(1-7) are not receptor mediated. Further, we found differences between Akr cells and Crl cells that are consistent with their genetic heterogeneity.

Testosterone increases blood pressure and cardiovascular and renal pathology in spontaneously hypertensive rats.

The objective of this paper was to test the hypothesis that testosterone (T) raises blood pressure (BP), which is associated with increased coronary adventitial collagen, whereas the hemodynamic force of BP increases the coronary media:lumen ratio. Five treatment groups of spontaneously hypertensive rat (SHR) were established (n = 8-10 per group): controls; hydralazine (HYZ); castration; castration + HYZ; and castration + HYZ + T + captopril. At 12 weeks of age, the castrate + HYZ group was divided so that the mean BP was the same in both groups (162 mmHg). Both groups continued to receive HYZ treatment; however one group received T implants. Also, at 12 weeks of age the castrate + HYZ + T + captopril group received T implants. BP in the HYZ group was reduced compared with controls (192 mmHg vs 218 mmHg, p < 0.01). Castration lowered BP to 170 mmHg (p < 0.01) compared with controls. However, T implants increased BP by 15 mmHg (p < 0.02) in the castrate + HYZ group and by 44 mmHg in the castrate + HYZ + captopril group (p < 0.01). Captopril in combination with HYZ significantly reduced BP compared with controls but T replacement increased BP and coronary collagen deposition in spite of HYZ and captopril treatment.

Determination of the prime electrostatic endothelial cell transplantation procedure for e-PTFE vascular prostheses.

The purpose of this study was to evaluate the extent of cellular adhesion (density and morphological maturation), cellular membrane damage, and cellular viability after an electrostatic transplantation of human umbilical vein endothelial cells (HUVECs) onto 6-cm segments of 4-mm I.D. e-PTFE (GORE-TEX) vascular prostheses using a prototype electrostatic endothelial cell transplantation device (EECTD). The electrostatic transplantation parameters evaluated were the apparatus-applied voltage and transplantation time. By our definition, the combination of applied voltage and transplantation time that met the a priori criteria of: 1) maximum transplanted cellular viability, 2) maximum transplantation density, 3) maximum morphological maturation (degree of cellular flattening), and 4) minimal cellular membrane damage would be the prime transplantation procedure. The results of the experimentation indicated that the prime conditions for HUVEC transplantation were obtained when +1.0 V was applied for a transplantation time of 16 min. These conditions achieved an average viable graft surface coverage of 97.4+/-1.6% with an average transplantation density of 73,540+/-8.514 HUVECs/cm2. Furthermore, the transplanted HUVECs were morphologically mature (flattened) with minimal apparent cellular membrane damage (lysis or pitting). The overall clinical significance of this study is that viable endothelial cell transplantation to synthetic vascular grafts can be accomplished at high cellular densities and morphological maturation in 16 min using the EECTD. With the promising in vitro transplantation results, the next logical investigations will include additional in vitro evaluations (cellular retention upon shear stress exposure and biochemical assays) followed by in vivo evaluations to examine thromboresistance and influence on intimal/anastomotic hyperplasia.

Review of the Y chromosome and hypertension.

The Y chromosome from spontaneously hypertensive rats (SHR) has a locus that raises blood pressure 20-25 mmHg. Associated with the SHR Y chromosome effect is a 4-week earlier pubertal rise of testosterone and dependence upon the androgen receptor for the full blood pressure effect. Several indices of enhanced sympathetic nervous system (SNS) activity are also associated with the SHR Y chromosome. Blockade of SNS outflow reduced the blood pressure effect. Salt sensitivity was increased by the Y chromosome as was salt appetite which was SNS dependent. A strong correlation (r = 0. 57, P<0.001) was demonstrable between plasma testosterone and angiotensin II. Coronary collagen increased with blood pressure and the presence of the SHR Y chromosome. A promising candidate gene for the Y effect is the Sry locus (testis determining factor), a transcription factor which may also have other functions.

Sodium intake is increased by social stress and the Y chromosome and reduced by clonidine.

The objectives were to determine 1) if female rats have higher Na intake than males and if social stress increases Na intake, 2) if the sympathetic nervous system (SNS) mediates the stress effects and the gender effect, and 3) if the Y chromosome (Yc) from a hypertensive father increases Na intake. Four rat strains (n = 10/group) of both sexes were used: 1) Wistar Kyoto normotensive (WKY), 2) an F(16) backcross with a Yc from a hypertensive father (SHR/y), 3) spontaneously hypertensive rat (SHR), and 4) an F(16) backcross with a Yc from a normotensive father (SHR/a). Females showed greater baseline Na intake than males (hypertensive strains), intruder stress increased Na intake, and clonidine decreased Na intake, but not in WKY or SHR females. SHR/y males had higher baseline Na intake compared with WKY males. In conclusion, the higher Na intake in females during baseline and stress was partially mediated through the SNS in hypertensive strains and the SHR Yc was partially responsible for the increased Na intake in SHR/y and SHR males compared with WKY.

Testosterone effects on renal norepinephrine content and release in rats with different Y chromosomes.

The Y chromosome in spontaneously hypertensive rats (SHR) and stroke-prone rats has been shown to contain a locus that contributes to the hypertensive effect; both the sympathetic nervous system and testosterone may be involved. The objective of this study was to look at the effects of testosterone on renal norepinephrine (NE) release and content in the isolated perfused kidney in different Y chromosome backgrounds. The study involved male SHR, Wistar-Kyoto rats (WKY), and 2 consomic strains with different Y chromosomes (n=5 to 8 per group). Adult animals were castrated, and implants containing testosterone propionate were placed at the base of the neck. Blood testosterone levels were measured by radioimmunoassay 2 weeks after castration. The left kidney was isolated and perfused with oxygenated Krebs solution at a constant flow and temperature with KCl and electrical stimulation of the renal nerves. Perfusate was collected and analyzed for NE by high-performance liquid chromatography. Lactate dehydrogenase analyses were performed as a marker for potential tissue damage. Renal perfusate and renal tissue NE levels were significantly elevated by testosterone. The average NE increase with a single testosterone implant was 13.2 ng/mL, and for a double testosterone implant it was 29.8 ng/mL. The Y chromosome from the SHR produced a significant increase in renal NE release compared with the WKY Y chromosome. Significance was shown between all groups: 1 versus 2 implants, P=0.0067; 1 versus sham implants, P=0.015; 2 versus sham implants, P<0.001. In conclusion, testosterone caused an enhanced renal NE release that was strain-specific, with the Y chromosome raising renal NE content and release.

Female Wistar-Kyoto and SHR/y rats have the same genotype but different patterns of expression of renin and angiotensinogen genes.

OBJECTIVES: To evaluate whether renin and angiotensinogen gene expression in females from two strains of rats that share the same autosomes and X chromosomes differs. Female SHR/y rats have the parental Wistar-Kyoto rat autosomes and X chromosomes and have no chromosomes of spontaneously hypertensive rat origin; thus they are genetically equivalent to female Wistar-Kyoto rats. DESIGN AND METHODS: Because these genes are regulated by steroid hormones, we investigated the effects of removal of estrogen (ovariectomy) and addition of androgen (testosterone implants) on three groups of female SHR/y rats and the parental rat strain Wistar-Kyoto rat with groups of intact (control) rats, rats subjected to ovariectomy at age 3 weeks, and rats subjected to ovariectomy with a testosterone implant at age 3 weeks. RESULTS: The combination of removing estrogen early in development and supplementing the ovariectomized females with testosterone revealed strain differences in response of blood pressure. Renin and angiotensinogen messenger RNA levels appear to be regulated coordinately within each strain, although actual levels of messenger RNA differ between the strains. CONCLUSIONS: Similar patterns of responses of renin and angiotensinogen genes to ovariectomy and ovariectomy plus testosterone suggest that regulation of the genes is likely to be similar or coordinate. Differences in regulation of renin-angiotensin system genes between strains may result from epigenetic mechanisms such as genome imprinting of these genes or of another gene that functions as a common regulator of renin and angiotensinogen.

In vitro evaluation of electrostatic endothelial cell transplantation onto 4 mm interior diameter expanded polytetrafluoroethylene grafts.

PURPOSE: To perform an in vitro evaluation of electrostatic endothelial cell transplantation of human umbilical vein endothelial cells (HUVEC) onto segments of 4 mm internal diameter expanded polytetrafluoroethylene (ePTFE) vascular prostheses. METHODS: This evaluation consisted of exposing vascular graft segments that had been subjected to either electrostatic or gravitation transplantation with HUVEC to a physiologic shear stress (15 dynes/cm2) under steady flow conditions within a flow loop system. Biochemical assays were performed on freshly transplanted grafts by means of radioimmunoassay for prostacyclin and thromboxane A2. RESULTS: There was a 30% loss of HUVEC after 30 minutes of shear stress exposure from the grafts subjected to gravitational transplantation with no additional significant (alpha = 0.05) loss after 120 minutes. Grafts subjected to electrostatic transplantation had no significant (alpha = 0.05) loss of HUVEC during exposure to physiologic shear stress. Furthermore, after 120 minutes of shear-stress exposure, the grafts subjected to electrostatic transplantation (78,420 +/- 6274 HUVEC/cm2) retained 2.3 times more HUVEC than the counterparts subjected to gravitational transplantation (34,427 +/- 4637 HUVEC/cm2). The biochemical assay results indicated no significant (alpha = 0.05) production of prostacyclin or thromboxane A2 regardless of the method of cell transplantation. CONCLUSIONS: (1) The electrostatic transplantation technique was superior to the gravitational transplantation technique in terms of cellular retention when the ePTFE grafts were exposed to physiologic shear stress. (2) Production of prostacyclin and thromboxane A2 did not differ between transplanted HUVEC subjected to gravitational or electrostatic procedures.

Spontaneously hypertensive rat Y chromosome increases indexes of sympathetic nervous system activity.

Previous studies from our laboratory have demonstrated that the Y chromosome from the spontaneously hypertensive rat (SHR) is responsible for a significant portion of the elevated blood pressure and also produces an earlier pubertal rise in plasma testosterone. We performed the following studies to determine whether the SHR Y chromosome raises blood pressure by sympathetic nervous system responses as measured by adrenal chromogranin A and plasma and tissue catecholamines. Male SHR from the University of Akron colony were studied from 5 to 20 weeks of age. Blood pressure was measured by tail-cuff, tail artery cannulation, and aortic telemetry (Data Sciences); acute (air stress) and chronic (territorial colony) social stressors were compared; blood was collected for determination of plasma catecholamines; and adrenal glands were analyzed at 15 weeks for catecholamines. Rats with the SHR Y chromosome had higher blood pressure and plasma norepinephrine than those with the normotensive Wistar-Kyoto (WKY) Y chromosome. However, the SHR Y chromosome did not significantly change responsiveness to acute or chronic stressors. Phentolamine and clonidine prevented the stress responses. Adrenal chromogranin A levels were elevated 37% and 40% and adrenal norepinephrine content 29% and 100% at 4 and 10 weeks of age, respectively, in rats with an SHR Y chromosome compared with WKY. Chemical sympathectomy normalized blood pressure in all strains and significantly reduced norepinephrine (36% to 41%) in all strains except in WKY, which already had a normal blood pressure. In conclusion, the SHR Y chromosome appears to increase the chronic sympathetic nervous system. A potential mechanism could be a Y locus that influences chronic sympathetic nervous system activity, which may reinforce neurohumoral factors and structural components of the vessel wall, accelerating the development of hypertension.

Differential regulation of angiotensinogen transcripts after renin infusion.

To investigate angiotensinogen regulation in high-renin hypertension, we infused porcine renin intravenously at either a low (4 mU/kg per hour, n = 6) or high (20 mU/kg per hour, n = 9) dose into male Sprague-Dawley rats (225 to 250 g) for 5 days using osmotic minipumps. Control rats received 0.9% NaCl. In renin-infused rats, mean arterial pressure and plasma renin activity were significantly elevated. Both low- and high-renin infusions lowered plasma angiotensinogen levels. Plasma angiotension II was elevated in rats given renin but reached statistical significance only at the higher dose. Angiotensinogen mRNA isolated from the liver, adrenal gland, kidney, and brain was measured by slot blot analysis. Both renin doses were associated with significant decreases in the levels of liver and hypothalamic angiotensinogen mRNA. In the medulla oblongata, angiotensinogen mRNA was reduced only by the higher renin dose. The lower dose increased angiotensinogen mRNA in the adrenal gland, and in kidney, angiotensinogen mRNA level was unchanged by renin infusion. Angiotensinogen mRNA visualized on Northern blots showed that the number of mRNA species in liver decreased from three in control rats to a single mRNA species after renin infusion. Tissue differences in the size of the major angiotensinogen mRNA species were also apparent. This, together with changes in the total hybridization signal of angiotensinogen mRNA in tissues, suggests that renin differentially affects the different angiotensinogen mRNA transcripts. Results of this study indicate that angiotensinogen gene expression is regulated not only by alterations in levels of circulating angiotensin II but also by other mechanisms, presently unidentified, that are activated by renin infusions.

The cDNA encoding canine dihydrolipoamide dehydrogenase contains multiple termination signals.

A 2288-bp cDNA sequence encoding dihydrolipoamide dehydrogenase (DLDH; dihydrolipoamide: NAD+ oxido-reductase; EC 1.8.1.4) was obtained by isolating a 1762-bp cDNA clone from a canine skeletal muscle library in the vector, lambda UNIZAP, combined with PCR amplification of the 5' end of the mRNA. The DLDH cDNA sequence contains a 49-bp G+C-rich 5'-untranslated region (UTR), followed by 1527 bp of coding region, and 695 bp of 3'-UTR preceding a 17-bp poly(A) tail. The single open reading frame encodes a precursor DLDH of 509 amino acids (aa) that begins with a 35-aa leader sequence. The 3'-UTR includes six possible polyadenylation signals (three AATAAA, one TATAAA and two AATGAA) and one potential stem-loop region extending from bp 1969-1991. Alignment studies of the canine and human DLDH demonstrate homology within the coding region of 98% at the aa level and 94% at the nt level. Northern blot analysis using the cDNA clone as probe showed wide tissue distribution of the mRNA, with differences in the level of expression among tissues and possible utilization of different polyadenylation sites.

Tissue renin-angiotensin systems in renal hypertension.

Angiotensinogen messenger RNA (mRNA) levels were measured in the brain (hypothalamus, lower brain stem, cerebellum), liver, kidneys, and adrenal glands of rats made hypertensive by ligation of the aorta between the renal arteries. We also measured renin mRNA in the kidneys of these renal hypertensive rats. The early phase of hypertension (day 6) was associated with significant increases in plasma renin activity and levels of circulating angiotensin II. The circulating renin-angiotensin system was not activated in the later phase of hypertension (day 24). Angiotensinogen mRNA levels were elevated in the lower brain stem of hypertensive rats at both stages of hypertension. In contrast, angiotensinogen mRNA levels in the hypothalamus were increased only at day 6 after aortic ligation. Decreased levels of angiotensinogen mRNA were observed in the cerebellum in both the early and later phases of the hypertension. Angiotensinogen mRNA levels in the adrenal gland below the ligature fell in the early phases but rose in the later phases of hypertension. Renin mRNA levels of the ischemic kidney remained elevated at both the early and later phases, whereas in both ischemic and nonischemic kidneys, levels of angiotensinogen mRNA remained below sham values throughout the period of study. These results indicate differential expression of renin-angiotensin system mRNAs in tissues of renal hypertensive rats. The differential changes in the expression of angiotensinogen mRNA over the course of development and maintenance of renal hypertension suggest that factors in addition to angiotensin II are important in modulating the expression of renin-angiotensin system genes.

Peripheral and central angiotensin II regulates expression of genes of the renin-angiotensin system.

We investigated whether angiotensin (ANG) II has the potential to regulate expression of genes of the renin-angiotensin system (RAS) in peripheral and central tissues. ANG II (0.1 or 6.0 nmol/h) was infused by osmotic minipump into male Sprague-Dawley rats (225-250 g) for 5 days, either intravenously or intracerebroventricularly. We measured angiotensinogen mRNA in liver, adrenal glands, and brain (hypothalamus and lower brain stem), renin mRNA in the kidney, and angiotensin-converting enzyme (ACE) mRNA in the lung and testis by Northern blot analysis. We demonstrated that plasma ANG II increases the levels of liver angiotensinogen mRNA, decreases kidney renin mRNA, and decreases lung ACE mRNA. Intracerebroventricular administration of ANG II resulted in a different pattern of responses of the peripheral RAS components. Liver angiotensinogen mRNA was increased, and kidney renin mRNA was decreased by both doses of ANG II, whereas lung ACE mRNA remained unresponsive at either dose. Centrally mediated influences of ANG II are most likely indirect since plasma ANG II concentration was not changed. This study has revealed that ANG II has profound diverse effects that influence the regulation of its formation. Further, results indicate that genes of the RAS responded to exogenous ANG II in both tissue- and route-specific ways.

Angiotensin-(1-7): a new hormone of the angiotensin system.

We provide a new foundation for an alternative interpretation of the biochemical physiology of the brain and other tissue angiotensin systems on the basis of research done in our laboratory. This perspective is prompted by the discovery that angiotensin-(1-7) has cellular functions that differ from those established for angiotensin II. Although angiotensin-(1-7) is not an agonist in terms of activating vasoconstriction, stimulating thirst, or promoting aldosterone release, the heptapeptide caused neuronal excitation and vasopressin release with a potency similar to that found with angiotensin II. Furthermore, angiotensin-(1-7) enhances the production of prostanoids by a receptor-mediated event that causes no associated rise in intracellular Ca2+. These actions of angiotensin-(1-7) provide a new understanding of the heterogeneous functions of angiotensin peptides as modulators of a wide range of regulatory functions in mammals.

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA.

We have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen has not previously been available, Expression noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because greater than 99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing us to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

The transcriptional response of the human chorionic gonadotropin beta-subunit gene to cAMP is cycloheximide sensitive and is mediated by cis-acting sequences different from that found in the alpha-subunit gene.

Cyclic AMP stimulates transcription of the genes encoding the alpha- and beta-subunits of human CG (hCG). Although the cis-acting cAMP response element (CRE) of the alpha-subunit gene has been extensively characterized, relatively little is known about the putative response element of the hCG beta gene. Here, we demonstrate that cAMP stimulation of hCG beta gene transcription requires ongoing protein synthesis, whereas cAMP stimulation of alpha-subunit gene transcription does not. These results suggest that cAMP-stimulated transcription of the alpha and hCG beta genes may involve different cis-acting elements and trans-acting factors. Accordingly, we have constructed a series of deletion mutants consisting of fragments of the hCG beta promoter-regulatory region linked to the bacterial chloramphenicol acetyl transferase gene. To potentiate detection of a CRE, we constructed vectors containing the Rous sarcoma virus enhancer to augment transcriptional activity of the hCG beta-promoter. We also used vectors designed to determine whether regions containing a putative CG beta CRE could confer cAMP responsiveness to a heterologous promoter. These constructs were evaluated for activity by transfection into choriocarcinoma (BeWo) cells. Transient expression of these vectors identifies a broad region which renders transcription of the chloramphenicol acetyl transferase gene responsive to cAMP. Surprisingly, division of the hCG beta 5'-flanking and untranslated regions into smaller fragments led to loss of cAMP responsiveness, suggesting that the hCG beta CRE may be comprised of multiple domains dispersed across the proximal 650 base pairs of CG beta 5'-flanking and untranslated sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP and phorbol esters interact synergistically to regulate expression of the chorionic gonadotropin genes.

Previous studies have shown that activators of the protein kinase A pathway increase transcription of the genes encoding the alpha- and beta-subunits of human chorionic gonadotropin (hCG) in choriocarcinoma cell lines. Here, we show that treatment of choriocarcinoma cells with activators of protein kinase C, such as phorbol myristate acetate (PMA) and dioctanoylglycerol, increases accumulation of the mRNAs for both subunits of hCG by 3-4-fold. In contrast, a phorbol ester which fails to activate protein kinase C, phorbol 12 beta,13 alpha-didecanoate, has no effect on hCG mRNA levels. To test the possibility that these two major intracellular signaling pathways interact, we treated choriocarcinoma cells with PMA, forskolin, or PMA and forskolin together. Treatment with either agent led to a 2-3-fold increase in hCG mRNA levels, whereas treatment with both agents resulted in a 9-fold increase. This synergistic response also occurred when choriocarcinoma cells were treated with PMA and 8-Br-cAMP. Furthermore, PMA did not increase intracellular cAMP levels, suggesting that these two pathways interact subsequent to cAMP generation. PMA also increased transcription of the hCG alpha- and beta-genes by 2-3-fold. Whereas transcription of the alpha subunit gene increases synergistically after treatment with both PMA and forskolin, transcription of the hCG beta-gene was limited to the increase caused by either agent alone. This latter result suggests that regulation of hCG beta mRNA accumulation is more complex than that of alpha-subunit mRNA and probably involves both transcriptional and post-transcriptional components.