Cyclic Nucleotide Modulation of Herpes Simplex Virus Latency and Reactivation

Transcription

1 Investigative Ophthalmology & Visual Science, Vol. 30, No. 10, October 1989 Copyright Association for Research in Vision and Ophthalmology Cyclic Nucleotide Modulation of Herpes Simplex Virus Latency and Reactivation Moire Sainz de la Maza, Peter A. Wells, and C. Srephen Fosrer Clinical observations and experimental studies suggested that the relative proportions of ganglionic neuronal intracellular cyclic adenosine monophosphate (c-amp) and cyclic guanosine monophosphate (c-gmp) concentrations may influence the state or activity of herpes simplex viral DNA in its relationship with the host cell DNA. We studied the effects of putative modulators of intracellular cyclic nucleotide levels on herpes simplex virus (HSV) reactivation from latency in murine trigeminal ganglion cells. We also investigated the effects of these same mediators on the c-gmp and/or c-amp concentrations in HSV-latently infected trigeminal ganglion cells and in acyclovir-suppressed, HSVinfected neuroblastoma cells. Cholera toxin and theophylline increased c-amp levels (2-fold and 5-fold at 1 min and 30 sec, respectively for cholera toxin and 2-fold and 1.5-fold at 1 min and 30 sec for theophylline) and enhanced the rapidity of HSV reactivation from latency (). Exogenous dibutyryl c-amp also stimulated viral reactivation (). increased c-gmp levels (7-fold and 6-fold at 15 sec and 30 sec, respectively), produced no significant change in c-amp levels, and delayed HSV reactivation from latency (). None of these mediators had a demonstrable effect on HSV replication. Invest Ophthalmol Vis Sci 30: ,1989 Recurrent herpetic keratitis remains a major cause of corneal blindness in developed countries. 1 Following primary infection with herpes simplex virus 1 (HSV-1), the viral DNA persists within the neurons of sensory ganglia in a special relationship, the exact molecular details of which are incompletely understood. 2 " 4 These "latently" infected ganglia, including the sensory neurons for the eye, have been shown to harbor HSV DNA even in individuals who had no history of herpes eye infection. A substantial body of evidence suggests that latent HSV infections may become manifest periodically; corneal scarring results primarily from reactivation of latent viral DNA, viral assembly and replication, and reestablishment of a productive infection within the cornea, with the attendant host inflammatory response. Current therapy and experimental attempts to develop new antiviral agents have proven incapable of eradicating latent viral DNA from its host. 5 " 9 An al- From the Hilles Immunology Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts. Supported in part by Fullbright "la Caixa" Grant (Dr. Sainz de la Maza) and by NIH Grant EY (Dr. Foster). Submitted for publication: November 28, 1988; accepted April 17, Reprint requests: C. Stephen Foster, MD, Hilles Immunology Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA ternative therapeutic strategy might include efforts to maintain latency and discourage episodes of viral reactivation. A wide variety of physiological stimuli (fever, trauma, sunburn, menses, stress) to patients and experimental stimuli (epinephrine, prostaglandins, ultraviolet light) to animal models can trigger herpetic reactivation and clinical disease. 10 " 16 These "triggers" seem to share at least one common characteristic: through the action of catecholamines (stress, trauma, epinephrine) and/or arachidonic acid metabolites (sunburn, fever, menses, trauma, prostaglandins, ultraviolet light), or first messengers, they could all influence levels of intracellular cyclic nucleotides, or second messengers, such as cyclic adenosine monophosphate (c-amp) or cyclic guanosine monophosphate (c-gmp). We hypothesized that the relative proportions of intracellular c-amp and c-gmp concentrations might influence the state of latency or reactivation of the herpes viral DNA; we have reported preliminary results supporting this hypothesis. 17 This report presents the results of detailed studies of the effects of putative modulators of intracellular cyclic nucleotide levels on HSV-1 reactivation from latency in a murine trigeminal ganglion model and the correllative effects of these mediators on c-gmp and c-amp concentrations in murine trigeminal ganglion and in neuroblastoma cells. Development of a murine neuroblastoma cell culture model 2154

2 No. 10 CYCLIC NUCLE0TIDE5 AND LATENT HERPES / Sainz de la Maza er ol 2155 for further study of the relationship between c-amp/ c-gmp concentrations and HSV latency will be the subject of a future communication. Cell Culture Material and Methods Monkey kidney Vero cell monolayers (CCL 81, American Type Culture Collection, Rockville, MD) for HSV assay were maintained in 75 cm 2 flasks (Falcon Plastics, Fisher Scientific Inc., Pittsburgh, PA) using Minimum Essential Medium (MEM) (Gibco, Grand Island, NY) with Earle's salts containing 5% mg/ml heat-inactivated fetal bovine serum (Gibco), 0.58 mg/ml L-glutamine (Gibco), 25 g/ml Fungizone (Flow Laboratories, McLean, VA), 100 U/ml penicillin and 100 g/ml streptomycin (Gibco). Confluent Vero cell monolayers were trypsinized and plated into 12-well 4.5 cm 2 tissue culture plates (Linbro, Flow Laboratories). Cultures were incubated in a humidified atmosphere of 95% air and 5% CO 2. Mouse neuroblastoma C-1300 cell monolayers (CCL 147, American Type Culture Collection) were maintained in 75 cm 2 flasks using Ham's F-12 nutrient mixture with L-glutamine (Gibco) supplemented with 5% mg/ml heat-inactivated fetal bovine serum, 25 g/ml Fungizone, 100 U/ml penicillin and 100 g/ml streptomycin. Confluent neuroblastoma cell monolayers were trypsinized and plated into 6- well, 9.62 cm 2 tissue culture plates. Cultures were incubated in a humidified atmosphere of 95% air and 5% CO 2 in air. Virus Stock HSV-1, strain KOS, was obtained from Dr. David Knipe (Harvard Medical School, Boston, MA) and grown in our laboratory by passage in Vero cell monolayers. 17 " 19 KOS-strain-infected Vero cell monolayers were harvested when a 4+ cytopathic effect was observed (all cells infected and detached). The infected cells were freeze-thawed three times and centrifuged at 1500 g. Aliquots of the supernatants, stored at 70 C were selected at random for dilution and standard plaque assay on Vero cells. 20 Each assay was repeated at least three times. Animals Female A/J mice, 5 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in a laminar flow microisolation environment. All studies presented adhered to the 1983 ARVO Resolution on the Use of Animals in Research. Mediators Cholera toxin, type Inaba, was purchased from Calbiochem, Behring Diagnostics (La Jolla, CA); dibutyryl c-amp from Sigma (St. Louis, MO); carbamylcholine (Isopto-Carbachol) from Alcon (Puerto Rico); and theophylline (Aminophylline) from Elkins-Sinn, Inc. (Cherry Hill, NJ). In Vivo Ganglionic Latent Viral Infection Following an eight-scratch corneal abrasion with a 25-gauge needle, the right corneas of 60 A/J mice were inoculated with 4 X 10 4 PFU HSV-1 in 5 fi\ of MEM. Clinical evaluation of lids and cornea one week later confirmed viral infection. Ipsilateral trigeminal ganglia were aseptically removed 28 days after inoculation, rinsed three times in MEM containing antibiotics and cultivated individually on confluent Vero cell monolayers. Ten ganglia were homogenized individually with a Pyrex tissue Grinder in 1 ml of media, were put through three freeze-thaw cycles and were centrifuged at 500 g. The supernatants from each individual ganglion were placed on Vero cell monolayers to assay for the presence of mature, infectious virions. Ten control (medium only) latently infected ganglia were maintained in organ culture for 21 days. The 40 remaining latently infected ganglia were divided into four groups of ten ganglia each. Putative modulators of intracellular cyclic nucleotide concentrations were added to the culture medium of each of these four groups in the following concentrations: cholera toxin, 0.1 g/ml; carbamylcholine, 1 mm; dibutyryl c-amp, 1 mm; theophylline, 0.1 mm. Media from all six groups were changed daily, and in the four experimental (mediator-treated) groups, fresh mediator was added each day, to produce final concentrations shown above. Cultures were maintained for 21 days with MEM + 5% FCS at 37 C in a humidified 95% air and 5% CO 2 atmosphere. The Vero cell monolayers were observed daily for characteristic herpes simplex-induced cytopathic effects (CPE); 100% of control ganglia showed a positive CPE by day 21. In Vitro Neuroblastoma Cell Viral Replication Confluent mouse neuroblastoma cell cultures were infected for 1 hr with KOS strain HSV-1 (MOI = 1) and were then cultured without (control) or with each of the four mediators for 24 hr. Preliminary dose-response studies (unreported) determined both the limits of mediator concentration due to toxicity and the optimal concentrations producing unequivocal, reproducible effects of the intracellular cyclic nucleo-

3 2156 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1989 Vol. 30 tide concentrations. The cells from the six wells for each of the five groups were harvested and homogenized; after three freeze-thaw cycles and centrifugation at 500 g, aliquots of the supernatants underwent our standard blinded plaque assay for infectious virus on Vero cell monolayers. In Vitro Neuroblastoma Cell HSV-Infected, ACV-Suppressed Model The HSV-suppressed model was defined as HSV DNA positivity (shown by c-dna probing in situ hybridization), with concurrent negativity for mature virions as detected by antibody probing for gamma glycoproteins and by cocultivation of cell culture supernatant on Vero cell monolayers for detection of classic virus-induced cytopathic effect. The model was produced by incubating neuroblastoma cell monolayers for 1 hr with KOS strain HSV-1 (MOI = 1), removing the virus-containing medium, washing three times, and incubation with medium containing 20 ng/m\ of acyclovir. The degree of HSV gamma protein suppression in this model with the concentration of acyclovir employed was 100%. The neuroblastoma cells were viable during this HSVsuppressed infection model for up to 10 days; growth of the cultures to confluency, with subsequent cell death occurred beyond this point. Reactivation from "latency" or the suppressed state was defined by the appearance of infectious virions in the cell culture supernatant as detected by plaque assay on Vero cell monolayers. Further details of this model and results of viral latency-reactivation in it are the subjects of a publication currently in progress. The purpose of this description of the model in this report is to provide adequate background for the basis of our studies of the influence of the various mediators on intracellular cyclic nucleotide levels in this model. The experiment was replicated to confirm the accuracy of the initial results. c-amp Radioimmunoassay in Trigeminal Ganglia Ipsilateral trigeminal ganglia were aseptically removed from 39 A/J mice 28 days after corneal inoculation with KOS strain HSV-1 and incubated with MEM + 5% FCS for 16 hr. Different mediators were added to the individual ganglia for 30 sec or for 1 min. Eight ganglia were exposed to cholera toxin (0.1 g/ml) for 60 sec; six were incubated with this mediator for 30 sec. Nine ganglia were incubated with theophylline (0.1 mm) for 60 sec; eight were exposed to this mediator for 30 sec. Eight ganglia served as controls. Ganglia were washed 10 seconds with Hank's balanced salt solution (Gibco), frozen with liquid nitrogen and homogenized with cold 6% trichloroacetic acid. A competitive radioimmunoassay for c-amp (Rianen [ 125 I] c-amp radioimmunoassay kit, NEK-033, Dupont New England Nuclear Research Products, Boston, MA) was used to determine the amount of c-amp in each ganglion. Protein content of each ganglion was measured using a dye-binding assay (Bio-Rad protein assay, Bio-Rad Laboratories, Richmond, CA), and results were obtained by a linear regression method. c-amp and c-gmp Radioimmunoassay in Neuroblastoma Cell Cultures Forty-two confluent neuroblastoma cell cultures were infected for 1 hr with KOS strain HSV-1 (MOI = 1), washed three times with Ham's F-12 nutrient mixture, and maintained for 7 days with Ham's mixture plus 5% FCS with or without 1 ml of acyclovir 20 ^g/ml (Burroughs Wellcome Co., Research Triangle Park, NC) and/or 0.5 ml of neural growth factor 50 ng/ml (7S-NGF, Collaborative Research, Inc., Bedford, MA). Three cultures received medium alone, three received acyclovir, three received neural growth factor, and three received both acyclovir and neural growth factor. Media were changed every day. Each 6-well tissue culture plate was washed three times with Dulbecco's Modified Eagle Medium minus NaHCO 2 (Gibco) with 25 mm Hepes buffer adjusted to ph 7.4 with NaOH. Reactions to carbamylcholine were initiated by the addition of 1.5 ml of a solution containing this mediator dissolved in Dulbecco's Phosphate Buffered Saline (Gibco) for 15 sec, 30 sec or 1 min (five acyclovir-suppressed HSV-infected neural growth factor supported cultures for each time period). The carbamylcholine or medium was removed by aspiration, and cold 6% trichloroacetic acid was added to the monolayer to terminate the reaction. Five acyclovir-suppressed, HSV-infected neural growth factor-supported cultures not containing carbamylcholine served as controls for each of the three incubation periods (15 control cultures). A rubber policeman was used to scrape the cells from each well. The cells were sonicated and subjected to competitive radioimmunoassay for c-gmp. Forty-two additional neuroblastoma cell cultures were processed identically as described above and were assayed for c-amp. Protein content of the cells was measured as described above. Statistical Analysis The data were analyzed for differences between mean values using the student t-test.

4 No. 10 CYCLIC NUCLEOTIDES AND LATENT HERPES / Soinz de IQ MQZO er ol 2157 Table 1. Effect of mediators on a murine trigeminal ganglion of HSV-1 latency model Mediator Reactivation time* Difference from controls (Significance) Cholera toxin (0.1 /xg/ml) (1 mm) Dibutyrylc-AMP(1 mm) Theophylline (0.1 mm) Control (no mediators) Homogenization 7.6 ± ± ± ± ±0.447 no infectious virus decrease increase decrease decrease () () () () Mean number of days for 10 cultures + SEM. Results The effects of the different mediators on the amount of time to HSV reactivation from latency in the trigeminal ganglion organ cultures are shown in Table 1. Cholera toxin and theophylline significantly decreased the interval before HSV reactivation, compared with control cocultures without mediators (P < 0.005). Dibutyryl c-amp also decreased the time for viral reactivation (). Conversely, carbamylcholine produced longer intervals of latency, significantly retarding viral reactivation (). Homogenized ganglia were negative for 21 days of cocultivation, demonstrating the functional latency of the model. All co-cultured ganglia produced viral CPE on the Vero cells within 21 days of establishment of the cultures. Cholera toxin, carbamylcholine, dibutyryl c-amp and theophylline had no effect on HSV-1 replication in mouse neuroblastoma HSV-infected cells (Table 2). Table 3 shows the changes in the intracellular c-amp concentrations produced by cholera toxin and by theophylline in latently infected trigeminal ganglia. Cholera toxin increased c-amp levels 2-fold and 5-fold at 1 min and at 30 sec, respectively, indicating that the hypothesized intracellular molecular effect of adding this mediator to the cultures, namely increased c-amp levels, in fact occurs reproducibly and predictably. Theophylline also elevated c-amp levels as predicted. Neither acyclovir nor neural growth factor, nor a combination of the two added daily during 7 days to Table 2. Effect of mediators on HSV-1 replication in mouse neuroblastoma cells Groups (n = 6) Control (no mediators) Cholera toxin (0.1 n%/m\) (1 mm) Dibutyryl c-amp (1 mm) Theophylline (0.1 mm) Experiment 1, PFU X Experiment 2 PFU X HSV infected in neuroblastoma cell cultures produced a significant change in intracellular c-gmp levels (Table 4). When carbamylcholine was added to HSV-infected neuroblastoma cell cultures with acyclovir suppression of productive replication, intracellular c-gmp levels rose 7-fold and 6-fold after incubation for 15 sec and for 30 sec, respectively, but fell to normal levels at 1 min. These results are summarized in Table 5. Acyclovir and neural growth factor added daily during 7 days to HSV-infected neuroblastoma cell cultures did not produce detectable, significant changes in intracellular c-amp concentrations (Table 6). The addition of carbamylcholine to the acyclovirsuppressed, HSV-infected neuroblastoma cell model produced no significant effect on intracellular c-amp concentrations, even at the maximal concentration of carbamylcholine employable without production of toxic effects to the neuroblastoma cells (Table 7). Discussion In spite of the major recent advances in characterization of the viral genome and its relationship to host cell DNA during latent herpes simplex virus infection, the prospects for the development of "genetic surgery" or other molecular-level strategies capable of Table 3. Effect of cholera toxin and of theophilline on intracellular c-amp levels in HSV-latently infected trigeminal ganglia Mediator* Cholera toxin 1 min Cholera toxin 30 sec Theophylline 1 min Theophylline 30 sec Control 1 min n c-amp Concentration (picomoles/ng protein)^ ± ± ± ± ± 1.51 * Cholera toxin, 0.1 Mg/ml; theophylline, 0.1 mm. t Mean picomole + SEM corrected for % recovery. Difference from controls /><0.01

5 2158 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Ocrober 1989 Vol. 30 Table 4. Effect of acyclovir and of neural growth factor on HSV-infected neuroblastoma cells Table 6. Effect of acyclovir and of neural growth factor on HSV-infected neuroblastoma cells Group (n = 6)-\ c-gmp Concentration (picomoles/ng protein)* Group (n = 9)* c-amp Concentration (picomoles/\ig protein)^ Control 5.28 ± 0.53 Acyclovir, 20 /ig/ml 4.14 ± 0.39 NS Neural growth factor, 50 ng/ml 4.41 ±0.45 NS Acyclovir + neural growth factor 5.42 ± 0.35 NS Control Acyclovir 20 Mg/ m l Neural growth factor 50 ng/ml Acyclovir + neural growth factor rt ±5.75 NS =t 7.11 NS ±5.25 NS * Mean + SEM corrected for % recovery. t Two sample replicates from each of the three individual cultures. * Three sample replicates from each of three individual cultures, t Mean = SEM corrected for % recovery. Table 5. Effect of carbamylcholine on intracellular GMP levels in acyclovir-suppressed, HSV-infected neuroblastoma cells c-gmp Concentration Mediator* n = /5f (picomoles/ng protein)% 15 sec Medium 15 sec 30 sec Medium 30 sec 1 min Medium 1 min ± ± ±0.61' ± ± NS *, 0.05 mm. f Three sample replicates from each of the five individual cultures. i Mean ± SEM corrected for % recovery. eradicating viral DNA from its latent state in the neuron are very bleak. An alternative could be to identify and manipulate factors involved in the viral reactivation from the latent state. Clinical and experimental observations of well known provocateurs of reactivation from latency led us to hypothesize that the relative proportions of intracellular c-amp and c-gmp concentrations might influence the state or activity of the herpes viral DNA in its relationship with host cell DNA and hence might influence maintenance of or reactivation from latency. In vitro models of HSV latency, such as murine trigeminal ganglion and murine neuroblastoma cells, offer the means to explore physiological changes produced by some neurotransmitters and their effects on maintenance of latency or induction of reactivation Neuronal cell membrane receptors for neurotransmitters form oligomeric complexes with GTP-regulatory proteins. Control of cell membrane-associated enzymes, such as adenylate cyclase, resides in this receptor-regulatory protein coupling Cholera toxin binds to the stimulatory nucleotide regulatory unit (Ns), forming GTP-Ns, which activates adenylate cyclase. 22 ' 23 The adenylate cyclase catalizes the conversion of ATP to c-amp, resulting in elevated intracellular c-amp levels. Theophylline, a phosphodiesterase inhibitor, also increases intracellular c-amp levels by inhibiting the catabolic hydrolysis of c-amp catalyzed by phosphodiesterase. 24 The fact that both mediators increased c-amp levels in an HSV-latently infected trigeminal ganglion model and also accelerated HSV reactivation from latency indicates a role for elevated intracellular c-amp levels in promoting HSV reactivation from latency. Exogenous dibutyryl c-amp also stimulated viral reactivation in this model., an activator of muscarinic acetylcholine receptors, has been shown to increase intracellular c-gmp concentrations while producing no change or a slight decrease in intracellular c-amp levels. 25 This latter effect is secondary to adenylate cyclase inhibition via GTP-inhibitory nucleotide regulatory unit binding. These effects of carbamylcholine were confirmed in our acyclovir-suppressed, HSV-infected neuroblastoma cell culture model; furthermore, carbamylcholine retarded HSV reactivation from latency in our latently infected trigeminal ganglion model, indicating that elevated intracellular Table 7. Effect of carbamylcholine on intracellular AMP level in acyclovir-suppressed, HSV-infected neuroblastoma cells c-amp Concentration Mediator* nf (picomoles/^g protein)% 15 sec Medium 15 sec 30 sec Medium 30 sec 1 min Medium 1 min ± ± ± ± ± ±4.98 t Replicate samples from each of the five individual cultures; where n is less than 15 (three samples each culture), samples were lost in processing. t Mean ± SEM corrected for % recovery.

6 No. 10 CYCLIC NUCLEOTIDES AND LATENT HERPES / Soinz de lo Mozo er ol 2159 c-gmp levels may play a role in inhibiting HSV reactivation from latency. None of these mediators had a demonstrable effect on HSV-1 replication, indicating that the results presented above were related specifically to the phenomena of viral latency and reactivation. These findings may have profound clinical implications for patients with recurrent ocular herpes. Additional studies on a molecular level are in progress. Key words: cyclic nucleotides, HSV-1, viral reactivation, trigeminal ganglia, neuroblastoma cells References 1. Dawson CR and Togni B: Herpes simplex eye infections: Clinical manifestations, pathogenesis and management. Surv Ophthalmol 21:121, Cook ML, Bastone VB, and Stevens JG: Evidence that neurons harbor latent herpes simplex virus. Infect Immun 9:246, McLennan JL and Darby G: Herpes simplex virus latency: The cellular location of virus in dorsal root ganglia and the fate of the infected cell following virus activation. J Gen Virol 51:233, Stevens JG: Latent herpes simplex virus and the nervous system. Curr Top Microbiol Immunol 70:31, Field HJ and De Clercq E: Effects of oral treatment with acyclovir and bromovinyldeoxyuridine on the establishment and maintenance of latent herpes simplex virus infection in mice. J Gen Virol 56:259, Klein RJ, DeStefano E, Friedman-Kien AE, and Brady E: Effect of acyclovir on latent herpes simplex virus infections in trigeminal ganglia of mice. Antimicrob Agents Chemother 19:937, Pavan-Langston DN, Park NH, and Hettinger M: Ganglionic herpes simplex and systemic acyclovir. Arch Ophthalmol 99:1417, Trousdale MD, Dunkel EC, and Nesburn AB: Effect of acyclovir on acute and latent herpes simplex virus infection in the rabbit. Invest Ophthalmol Vis Sci 19:1336, Wohlenberg C, Openshaw H, and Notkins AL: In vitro system for studying the efficacy of antiviral agents in preventing the reactivation of latent herpes simplex virus. Antimicrob Agents Chemother 15:625, Hill TJ, Blyth WA, and Harbour DA: Trauma to the skin causes recurrence of herpes simplex in the mouse. J Gen Virol 39:21, Green MT, Rosborough JP, and Dunkel EC: In vivo reactivation of herpes simplex virus in rabbit trigeminal ganglia: electrode model. Infect Immun 34:69, Willey DE, Trousdale MD, and Nesburn AB: Reactivation of murine latent HSV infection by epinephrine iontophoresis. Invest Ophthalmol Vis Sci 25:945, Hill JM, Rayfield MA, and Haruta Y: Strain specificity of spontaneous and adrenergically induced HSV-1 ocular reactivation in latently infected rabbits. Curr Eye Res 6:91, Kwon BS, Gangarosa LP, Burch KD, deback J, and Hill JM: Induction of ocular herpes virus shedding by iontophoresis of epinephrine into rabbit cornea. Invest Ophthalmol Vis Sci 21:442, Blyth WA, Hill TJ, Field HJ, and Harbour DA: Reactivation of herpes simplex virus infection by ultraviolet light and possible involvement of prostaglandins. J Gen Virol 33:547, Harbour DA, Hill TJ, and Blith WA: Recurrent herpes simplex in the mouse: inflammation in the skin and activation of virus in the ganglia following peripheral stimulation. J Gen Virol 64:1491, Foster CS, Opremcak EM, and Tolchin N: Evidence for the potential influence of cyclic nucleotides on maintenance of or reactivation from latency of herpes simplex virus in trigeminal ganglionic neurons. Acta Ophthalmol, in press. 18. Dunkel EC, Green MT, and Rosborough JP: Suppression of HSV-1 infection in trigeminal ganglion cells. Invest Ophthalmol Vis Sci 25:525, Vahlne A and Lycke E: Herpes simplex virus infection of mouse neuroblastoma cells. Proc Soc Exp Biol Med 156:82, Rodbell M: The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284:17, Matsuzawa H and Nirenberg M: Receptor-mediated shifts in c-gmp and c-amp levels in neuroblastoma cells. Proc Natl Acad Sci USA 72:3472, Spiegel AM and Downs RW: Guanine nucleotides: Key regulators of hormone receptor-adenylate cyclase interaction. Endocrin Rev 2:275, Kurose H, Katada T, Amano T, and Ui M: Specific uncoupling by islet-activating protein, Pertussis toxin, of negative signal transduction via adrenergic, cholinergic and opiate receptors in neuroblastoma and glyoma hybrid cells. J Biol Chem 258:4870, Rail TW: Central nervous system stimulants: The methylxantines. In The Pharmacological Basis of Therapeutics, Goodman AG, Goodman LS, Rail TW, and Murad F, editors. New York, MacMillan Publishing Company, 1985, pp George WJ, Poison JB, Toole AG, and Goldberg ND: Elevation of guanosine 3',5'-cyclic phosphate in rat heart after perfusion with acetylcholine. Proc Natl Acad Sci USA 66:398, 1970.