Transcriptional and post-transcriptional regulation of tyrosine hydroxylase messenger RNA in PC12 cells during persistent stimulation by VIP and PACAP38: differential regulation by protein kinase A and protein kinase C-dependent pathways.

VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration-response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6-38] blocked the effects of each peptide on TH mRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr(1)-D-Phe(2))-GRF-(1-29)-NH(2) was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absence of elevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion that VIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.

Effects of acute restraint stress on tyrosine hydroxylase mRNA expression in locus coeruleus of Wistar and Wistar-Kyoto rats.

Norepinephrine (NE) is thought to play a role in the stress response, and may be involved in stress-related psychopathological conditions such as depression or anxiety. Heterogeneity in individual responses to the same stressor suggest that a genetic susceptibility to the effects of stress may contribute to such pathology. To address possible mechanisms underlying this genetic aspect of the stress response, we examined acute stress-induced changes in mRNA expression for several components of the NE system in the locus coeruleus (LC) and adrenal medullae of stress-susceptible Wistar-Kyoto (WKY) rats and their parent Wistar (W) strain. Expression of tyrosine hydroxylase (TH), NE transporter (NET) and alpha(2A) receptor mRNA were measured in the LC by in situ hybridization 30 min and 2 h after the onset of 30 min restraint stress. Adrenal TH mRNA was measured by slot blots. No basal differences were observed for any measure, but in the LC, expression of TH mRNA increased by 40% in W rats at 30 min (n=8, p<0.05) and returned toward baseline by 2 h, while WKY rats showed only a non-significant 29% increase at 2 h. In contrast, adrenal TH mRNA expression increased in WKY rats at 2 h (n=3, p<0.05), with no significant change in W rats. NET and alpha(2A) mRNA were unaltered by restraint stress in both strains. Differences in the stress-reactivity of TH gene expression in the central and peripheral noradrenergic systems may be related to differences in behavioral coping strategies and autonomic responsivity to stress in these strains, and suggest that differences in noradrenergic reactivity may contribute to genetic susceptibility to stress-related pathology.

Transcriptional and posttranscriptional control of tyrosine hydroxylase gene expression during persistent stimulation of pituitary adenylate cyclase-activating polypeptide receptors on PC12 cells: regulation by protein kinase A-dependent and protein kinase A-independent pathways.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine release and biosynthesis in sympathetic postganglionic cells. Moreover, PACAP receptor activation in cultured adrenal chromaffin and superior cervical ganglion cells has been reported to increase the expression of the gene coding for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. However, the relative contribution of transcriptional and posttranscriptional mechanisms to the effects of PACAP on TH gene expression has not been evaluated. Therefore, in this study we compared the temporal effects of PACAP on TH gene transcription with the duration of its effects on TH mRNA levels. We had previously shown that vasoactive intestinal polypeptide, peptide histidine isoleucine, and secretin, peptides closely related to PACAP, induce TH gene expression through a cyclic AMP (cAMP)-dependent pathway. Therefore, using a mutant PC12 cell line deficient in cAMP-dependent protein kinase II (PKA), we also evaluated the role of the cAMP pathway in the effect of PACAP on TH gene expression. Continuous treatment of wild-type PC12 cells with PACAP (1 nM) increased TH mRNA levels maximally by 12 h and maintained TH mRNA at near maximal levels for at least 2 days. In contrast, the rate of TH gene transcription, as measured by a nuclear run-on assay, was maximal by 1 h and returned to basal levels by 3 h. The fact that a new steady-state level of TH mRNA was achieved and maintained for days in the absence of a sustained increase in TH gene transcription supports the involvement of posttranscriptional mechanisms. Removal of PACAP after 12 h, a time at which TH gene transcription was at basal levels, resulted in a subsequent return of TH mRNA to unstimulated levels within 36 h. Thus, continuous PACAP stimulation is required to maintain sustained increases in TH mRNA levels in the absence of a sustained elevation of transcription. To examine the role of the cAMP pathway in these effects, we compared the effects of PACAP in wild-type PC12 cells and in a mutant PC12 cell line (A126-1B2) that is deficient in PKA. PACAP failed to stimulate either TH mRNA levels or TH gene transcription in the mutant cells. In contrast to the effects of PACAP, dexamethasone increased TH mRNA levels by the same magnitude in both cell lines. It is noteworthy that stimulation of the PKA-deficient mutant cells with a combination of PACAP and dexamethasone (1 microM) produced a synergistic increase in TH mRNA levels, which was nearly twice that induced by dexamethasone stimulation alone. This synergistic effect was not transcriptionally mediated. The effect of the combined treatment on TH gene transcription was identical to the effect of dexamethasone alone. Taken together, these data indicate that PACAP regulates TH gene expression through a transcriptional mechanism requiring an intact cAMP pathway and through posttranscriptional mechanisms under the control of a cAMP-independent pathway(s).

Risperidone: clinical outcome predictors and cost-effectiveness in a naturalistic setting.

Although risperidone seems to be a safe and effective treatment for the management of psychotic symptoms, its acquisition cost is considerably higher than that of conventional antipsychotics, and its precise role in managing psychiatric illnesses has yet to be defined. The purpose of this investigation was to examine the relationship of patient demographic variables to therapeutic outcomes and to analyze the financial impact of risperidone on the treatment of psychotic symptoms. Subjects included in this 2-year, retrospective cohort, intent-to-treat analysis were all patients initiated on risperidone therapy at an inpatient psychiatric treatment facility. Clinical outcomes were assessed from the absolute change in hospitalized days, total number of psychotropic medications prescribed, and historic Clinical Global Impression severity scores. Logistic regression analysis was conducted to analyze the potential relationship to certain demographic variables to therapeutic response. The cost-benefit analysis compared the direct treatment costs incurred by the institution before and after risperidone initiation. Of the 66 patients originally started on risperidone, 57 completed a therapeutic trial. A clinical response was evident in 54 percent of these patients overall. Logistic regression analysis identified previous treatment intolerance and a negative history of substance abuse as predictive of therapeutic success with risperidone (p = .0006 and p = .01, respectively). Hospitalization rates declined by 43 percent among treatment responders and by 1.3 percent among nonresponders resulting in a net annual savings of $147,962. Risperidone may be efficacious in many patients who had previously failed antipsychotic trials. Patients who had been unable to tolerate traditional antipsychotics and those who lacked a documented history of substance abuse were uniquely responsive to risperidone treatment. The significant decline in hospitalized days that was observed among responsive patients seems to indicate that risperidone may be a cost-effective approach to the management of psychotic symptoms.

One thousand and six consecutive laparoscopic intraoperative cholangiograms.

Intraoperative cholangiography was successfully performed in 1,000 out of 1,006 attempts in 1019 consecutive cholecystectomies. There were 783 chronic, 95 acute, 61 fibrotic, 27 gangrenous and 40 cases of hydrops of the gallbladder in those laparoscopic cholecystectomies performed. unsuspected common duct stones were identified in 5% of the patients. There were no injuries resulting from intraoperative cholangiography performed via the cystic duct. In this large series, routine cholangiography was thought to be helpful in the prevention of common bile duct injuries and the establishment of abnormal anatomy. In non-acute cholecystitis, intraoperative cholangiography is necessary due to the importance of abnormal anatomy verification. The technique of laparoscopic cholecystectomy differs greatly from that of open technique, and, therefore, routine intraoperative cholangiography is strongly advised.

Expression and modulation of 5-hydroxytryptamine1A receptors in P11 cells.

P11 cells were transfected with DNA for the human 5-hydroxytryptamine1A (5-HT1A) receptor. These cells stably expressed the 5-HT1A receptor coupled to the inhibition of adenylyl cyclase, and not to the stimulation of phosphoinositide hydrolysis. Homologous and heterologous regulation of the 5-HT1A receptor was studied in this cell system. Pretreatment of P11-5HT1A cells with the 5-HT1 receptor agonist 5-carbox-amidotryptamine (5-CT) resulted in a 3-fold increase in both basal and forskolin-stimulated cAMP accumulation, and desensitization of the 5-HT1A receptor as indicated by a decrease in the potency of 8-hydroxydipropylaminotetralin (8-OH-DPAT) to inhibit forskolin-stimulated cAMP accumulation (vehicle-treated cells: EC50 = 2.3 +/- 0.8 nM; 5-CT-treated cells: 9.9 +/- 0.4 nM). The sensitization of adenylyl cyclase as a result of chronic agonist exposure was prevented by the 5-HT1A antagonist WAY100635, which indicated that the effect of 5-CT pretreatment on basal and forskolin-stimulated cAMP accumulation was mediated by 5-HT1A receptor activation. Pretreatment of cells with pertussis toxin abolished the inhibition of forskolin-stimulated cAMP accumulation mediated by 5-HT1A receptor activation and prevented the sensitization of adenylyl cyclase as a result of chronic 5-HT1A receptor agonist exposure. Pretreatment of P11-5HT1A cells with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), also resulted in desensitization of the 5-HT1A receptor, as indicated by a marked decrease in the potency and intrinsic activity of 8-OH-DPAT. No change in the binding characteristics (i.e., Kd or Bmax) of [3H]8-OH-DPAT to 5-HT1A receptor sites was observed after 5-CT or PMA treatments. Activation of alpha-1 adrenergic receptors, but not 5-HT2A receptors, had effects on 5-HT1A receptor responsiveness similar to those seen with PMA pretreatment. In P11-5HT1A cells, homologous regulation of the 5-HT1A receptor was characterized by sensitization of adenylyl cyclase and a decrease in agonist potency, whereas heterologous regulation of the 5-HT1A receptor was characterized by a greater decrease in agonist potency, as well as a marked decrease in intrinsic activity.

Reasons for early recurrence following laparoscopic hernioplasty.

The incidence and reasons for early recurrences following laparoscopic hernioplasty have not been studied. Because the incidence is small and the follow up is short, a multi-institutional study was performed among the pioneers in the field. The incidence figures were obtained by survey of surgeons who had significant experience (over 100 cases) and kept concurrent records. Fifty-four recurrences (1.7%) occurred after 3229 laparoscopic hernia repairs. There were 1944 transabdominal preperitoneal (TAPP) repairs with 19 recurrences (1%) and 578 preperitoneal repairs with no recurrences. There were 345 onlay mesh (IPOM) repairs with seven recurrences (2%), and 286 plug and patch repairs with 26 recurrences (9%). Simple closures were performed 76 times with two recurrences (2.6%). Fifty-seven patients (three cases were referred to the author without incidence data but complete records for analysis) had 60 recurrent hernias. Recurrences were noted, on average, 5.1 months postoperatively (range 0-30 months). The most common reason for recurrence was that the mesh was too small - 36 (60%). The mesh was never stapled in 19 instances (32%), and the hernia was never repaired in three cases (20%). The clips pulled through the tissue in six cases (8%), and in 10 cases (15%) the repair has not yet been undertaken because the etiology was unclear. There was more than one reason in 19 patients. Technical factors were responsible for nearly all recurrences.

Incidence of complications following laparoscopic hernioplasty.

Smaller individual series on the outcome of laparoscopic hernioplasty techniques have been reported. This study reports on the complications of 3,229 laparoscopic hernia repairs performed by the authors in 2,559 patients. The TAPP (transabdominal preperitoneal) technique was the most frequently performed: 1,944 (60%). The totally preperitoneal technique was performed 578 (18%) times. The IPOM (intraperitoneal onlay mesh) repair was performed 345 (11%) times. The plug-and-patch technique was used 286 (9%) times and simple closure of the hernia defect without mesh was used in 76 (2%) repairs. Overall, there were 336 (10%) complications: 17 (0.5%) major and 265 (8%) minor. There were 54 (1.6%) recurrences, with a mean follow-up of 22 months. The TAPP technique had 19 (1%) recurrences and 141 (7%) complications. There were four bowel obstructions in this subgroup from herniation of small bowel through the peritoneal closure and trocar sites. The totally preperitoneal technique had no recurrence and 60 (10%) complications. The IPOM group had 7 (2%) recurrences and 47 (14%) complications. The plug-and-patch technique had 26 (9%) recurrences and 24 (8%) complications. The simple closure of the internal ring had 2 (3%) recurrences and 10 (13%) complications. Laparoscopic hernioplasty is not without complications. Laparoscopic hernioplasty is not without complications. Training, experience, and attention to technique will prevent some of these complications.

Laparoscopic cholecystectomy with operative cholangiogram.

Intraoperative cholangiography was successfully performed in 99% of 525 consecutive laparoscopic cholecystectomies. There were 386 chronic, 54 acute, 44 fibrotic, 19 gangrenous, and 22 hydrops cases. Unsuspected common duct stones were identified in 5% of patients. Routine cholangiography to clarify the biliary anatomy, however, was of greater significance than the discovery of unsuspected common duct stones.

Laparoscopic herniorrhaphy. A preperitoneal tension-free approach.

At the present time laparoscopic herniorrhaphy appears to be successfully performed by utilizing a preperitoneal approach. The major indications are bilaterals hernia and the recurrent hernia. Other routine hernias may be done at the decision of the surgeon and patient depending upon the necessity of rapid return to work and desire to minimize pain. Utilizing this approach, the recurrence rate at the present time is less than 1%, which compares favorably with all other types of repair.

Transabdominal preperitoneal herniorrhaphy.

There have been multiple different approaches to laparoscopic herniorrhaphy with a tendency toward exposure of the preperitoneal space with placement of a prosthesis into this area. The transabdominal preperitoneal (TAPP) approach is a reasonably simple, safe repair, resulting in a less than 1% recurrence rate with minimal complications. This relatively pain-free herniorrhaphy allows rapid return to normal activity.

Complications of laparoscopic herniorrhaphy.

Anterior inguinal hernia repair is the second-most-commonly performed abdominal operation and has been associated with low morbidity and mortality rates. The principle of laparoscopy has been applied to this surgical problem in a series of 762 patients with 841 inguinal hernias. Four types of laparoscopic repairs were conducted: (1) high ligation of the indirect inguinal hernia sac and closure of the internal ring (87 patients with 89 hernias); (2) plug and patch of the internal ring (74 patients with 87 hernias); (3) transperitoneal suture repair of the transversalis fascia to the iliopubic tract or Cooper's ligament (28 patients with 30 hernias); and (4) placement of a large prosthesis over the myopectoneal orifice (563 patients with 635 hernias). These early results indicate that the overall complication rates were low, especially when a large prosthesis was used to reinforce the myopectoneal orifice. It is concluded that laparoscopic inguinal herniorrhaphy is a safe and effective procedure with which to manage this surgical problem.

Preliminary experience with laparoscopic-guided colectomy.

Laparoscopic-assisted colonic resection was attempted in 18 patients: 12 patients had surgery for ascending colon lesions, three underwent attempted left-sided colectomies, and three patients had low anterior resection. Three early patients required conversion to open laparotomy when the pathologic lesion could not be identified under laparoscopic guidance. In patients undergoing laparoscopic surgery, postoperative pain was markedly reduced. Oral intake was begun between the first and third postoperative days, and average hospital stay was 4 days. No operative morbidity or mortality occurred in this small group of patients.

Laparoscopic cholecystectomy with operative cholangiogram.

Intraoperative cholangiography was successfully performed in 98% of 300 consecutive laparoscopic cholecystectomies, including 290 chronic, 44 acute, 33 fibrotic, 16 gangrenous, and 17 hydropsed cases. Unsuspected common duct stones were identified in 16 patients (4%). The presence of two surgeons and the ability of routine cholangiography to clarify the biliary anatomy produced excellent results with no injuries to the common duct in this series.

Laparoscopic cholecystectomy: laser versus electrosurgery.

Laparoscopic cholecystectomy has been reported to be a safe and effective way to remove a diseased gallbladder with essentially no morbidity or mortality (1,2). This procedure was first introduced in Europe using electrosurgery, and later introduced in the United States using the laser (1). No clinical trial compares the laser to the standard electrosurgery method. In a review, as well as an ongoing study, morbidity, mortality, intraoperative procedures, indications, and cost effectiveness are considered in these two groups. Three hundred cases were reviewed with 150 patients in each group. There were no deaths or significant complications in either group. In the laser group, bleeding seemed slightly more excessive during removal of the gallbladder from the liver bed and required more time to control. The electrosurgery group required slightly less operative time and was less costly. The postoperative recovery time was the same in each group.

Laparoscopic herniorrhaphy.

Laparoscopic herniorrhaphy is compared with conventional herniorrhaphy in 20 patients, who underwent laparoscopic herniorrhaphy utilizing a Mersilene plug and patch graft, and high ligation of the neck of the sac with an Endo-GIA. Patients were pain-free and returned to normal activity the first postoperative day. There was one recurrent direct inguinal hernia in an indirect repair. Laparoscopic herniorrhaphy appears to be a safe, effective way to repair indirect inguinal hernias and certain direct inguinal hernias. There is a marked reduction of pain and rapid return to normal activity. The disadvantage of this procedure is the lack of long-term follow-up.