Safety of maintaining elective and emergency surgery during the COVID-19 pandemic with the introduction of a Protected Elective Surgical Unit (PESU): A cross-specialty evaluation of 30-day outcomes in 9,925 patients undergoing surgery in a University Health Board.

Background: The COVID-19 pandemic has caused unprecedented health care challenges mandating surgical service reconfiguration. Within our hospital, emergency and elective streams were separated and self-contained Protected Elective Surgical Units were developed to mitigate against infection-related morbidity. Aims of this study were to determine the risk of COVID-19 transmission and mortality and whether the development of Protected Elective Surgical Units can result in significant reduction in risk. Methods: A retrospective observational study of consecutive patients from 18 specialties undergoing elective or emergency surgery under general, spinal, or epidural anaesthetic over a 12-month study period was undertaken. Primary outcome measures were 30-day postoperative COVID-19 transmission rate and mortality. Secondary adjusted analyses were performed to ascertain hospital and Protected Elective Surgical Unit transmission rates. Results: Between 15 March 2020 and 14 March 2021, 9,925 patients underwent surgery: 6,464 (65.1%) elective, 5,116 (51.5%) female, and median age 57 (39-70). A total of 69.5% of all procedures were performed in Protected Elective Surgical Units. Overall, 30-day postoperative COVID-19 transmission was 2.8% (3.4% emergency vs 1.2% elective P < .001). Protected Elective Surgical Unit postoperative transmission was significantly lower than non-Protected Elective Surgical Unit (0.42% vs 3.2% P < .001), with an adjusted likely in-hospital Protected Elective Surgical Unit transmission of 0.04%. The 30-day all-cause mortality was 1.7% and was 14.6% in COVID-19-positive patients. COVID-19 infection, age > 70, male sex, American Society of Anesthesiologists grade > 2, and emergency surgery were all independently associated with mortality. Conclusion: This study has demonstrated that Protected Elective Surgical Units can facilitate high-volume elective surgical services throughout peaks of the COVID-19 pandemic while minimising viral transmission and mortality. However, mortality risk associated with perioperative COVID-19 infection remains high.

Assessing the accuracy of tympanometric evaluation of external auditory canal volume: a scientific study using an ear canal model.

Tympanometric evaluation is routinely used as part of the complete otological examination. During tympanometric examination, evaluation of middle ear pressure and ear canal volume is undertaken. Little is reported in relation to the accuracy and precision tympanometry evaluates external ear canal volume. This study examines the capability of the tympanometer to accurately evaluate external auditory canal volume in both simple and partially obstructed ear canal models and assesses its capability to be used in studies examining the effectiveness of cerumolytics. An ear canal model was designed using simple laboratory equipment, including a 5 ml calibrated clinical syringe (Becton Dickinson, Spain). The ear canal model was attached to the sensing probe of a Kamplex tympanometer (Interacoustics, Denmark). Three basic trials were undertaken: evaluation of the tympanometer in simple canal volume measurement, evaluation of the tympanometer in assessing canal volume with partial canal occlusion at different positions within the model, and evaluation of the tympanometer in assessing canal volume with varying degrees of canal occlusion. 1,290 individual test scenarios were completed over the three arms of the study. At volumes of 1.4 cm(3) or below, a perfect relationship was noted between the actual and tympanometric volumes in the simple model (Spearman's ρ = 1) with weakening degrees of agreement with increasing volume of the canal. Bland-Altman plotting confirmed the accuracy of this agreement. In the wax substitute models, tympanometry was observed to have a close relationship (Spearman's ρ > 0.99) with the actual volume present with worsening error above a volume of 1.4 cm(3). Bland-Altman plotting and precision calculations provided evidence of accuracy. Size and position of the wax substitute had no statistical effect on results [Wilcoxon rank-sum test (WRST) p > 0.99], nor did degree of partial obstruction (WRST p > 0.99). The Kamplex tympanometer accurately and precisely assesses ear canal volume in the scientific model in both simple and partially occluded wax substitute scenarios up to a volume of 1.4 cm(3). These findings suggest that the Kamplex tympanometer could be used as an effective objective tool in evaluating the efficacy of topical cerumolytics in both laboratory and human models.

Inhaled vs subcutaneous effects of a dual IL-4/IL-13 antagonist in a monkey model of asthma.

BACKGROUND: Pitrakinra is a recombinant protein derived from human interleukin-4 (IL-4) that binds to IL-4Ralpha and acts as a competitive antagonist of IL-4 and IL-13. The studies reported here compare the dose-ranging effects of pitrakinra on allergen-induced airway hyperresponsiveness (AHR) and airway eosinophilia when administered subcutaneously (s.c.) or by inhalation to the Ascaris suum-sensitive cynomolgus monkey for the purpose of elucidating the primary site of pitrakinra's anti-asthmatic action. METHODS: Airway responsiveness to inhaled methacholine and bronchoalveolar lavage cell composition was determined before and after three allergen exposures with a 1-week course of twice-daily (b.i.d.) s.c. or inhaled pitrakinra or placebo treatment. RESULTS: Treatment with s.c. pitrakinra significantly reduced allergen-induced AHR, with a maximum effect of a 2.8- to 3.8-fold increase in methacholine PC(100) relative to control (P < 0.05) observed at b.i.d. s.c. doses of 0.05-0.5 mg/kg. Inhaled pitrakinra also significantly reduced AHR with a similar maximum effect of a 2.8- to 3.2-fold increase in methacholine PC(100) relative to control (P < 0.05) at nominal b.i.d. doses of 3-100 mg. The maximal effect on AHR following inhalation was observed at a plasma concentration which exhibited no efficacy via the subcutaneous route. The effect of pitrakinra on lung eosinophilia was not statistically significant following either route of administration, although lung eosinophil count was reduced in all studies relative to control. CONCLUSION: Local administration of pitrakinra to the lung is sufficient to inhibit AHR, one of the cardinal features of asthma, indicating the therapeutic potential of inhaled pitrakinra in the treatment of atopic asthma.

External auditory canal exostoses in white water kayakers.

OBJECTIVE: In this study, the presence and severity of external auditory canal exostoses (EACE) in a group of white water kayakers related to the duration and intensity of kayaking and the number of ear infections reported were identified. DESIGN: A community-based volunteer cross-sectional study was completed with 92 kayakers (69 men, 23 women; mean (SD) age 29.3 (8.72) years) and 65 control volunteers (37 men, 28 women; mean (SD) age 36.9 (14.9) years). After exclusions, 269 individual ears were examined (154 kayakers and 115 controls). Participants completed a questionnaire and underwent otoscopic examination. Main outcome measures were the presence and severity of EACE, the duration and frequency of kayaking and self-reported ear infections. Exclusion criteria included other cold-water exposure, known ear pathology or poor visualisation of the auditory canal. RESULTS: The findings demonstrated that 69.5% of kayaker ears and 1.7% of the control group were found to have EACE. Severity of EACE was significantly associated with the duration (p<0.01) and frequency (p<0.05) of kayaking with 90.6% of kayakers that had participated for more than 10 years having evidence of EACE. A significant relationship also existed between the number of self-reported ear infections and the severity of EACE (p<0.01). CONCLUSION: There is a positive relationship between the duration and frequency of white water kayaking and the presence and severity of EACE and associated ear infections.

Allergen dose dependency of the early- and late-phase cutaneous response in the cynomolgus monkey.

BACKGROUND: Cutaneous administration of allergen provides a means to confirm an allergic status, investigate the pathogenesis of allergic diseases, and/or provide a mechanism to evaluate the benefit of new potential therapeutics. OBJECTIVE: Studies were performed to characterize the allergen-induced cutaneous early- and late-phase response (EPR and LPR) in the cynomolgus monkey. METHODS: Following intradermal injections of Ascaris suum allergen, the cutaneous weal and flare EPR was measured 15 min post-injection, and skin biopsies were collected at 8-24 h to determine the optimal time of LPR occurrence. Biopsies were analysed for epidermal and dermal inflammatory changes. RESULTS: The EPR was dose related with a reproducible, measurable response at 1 : 10 000 and maximal at a 1 : 100 allergen dilution. In contrast, the threshold dose required for a reproducible LPR was much greater requiring a dilution of 6 : 100, suggesting independent mechanisms for the EPR and LPR. The LPR 20 h post-allergen injection induced an inflammatory response in the upper and deep dermis. The response was characterized by a moderate perivascular to diffuse inflammation consisting of mononuclear cells, neutrophils and eosinophils. Dexamethasone, while having no effect on the EPR, reduced dermal inflammation (upper dermis, P=0.004; deep dermis, P=0.03). Similarly, dermal eosinophilia was also reduced (upper dermis, P<0.001; deep dermis, P=0.02). CONCLUSION: Collectively, the results indicate the dose dependency of the EPR and LPR. Furthermore, our observations indicate the value of the LPR response in the cynomolgus monkey to evaluate new therapeutics for the treatment of allergic diseases such as atopic dermatitis.

Specific cleavage of model recombination and repair intermediates by the yeast Rad1-Rad10 DNA endonuclease.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for both nucleotide excision repair and certain mitotic recombination events. Here, model recombination and repair intermediates were used to show that Rad1-Rad10-mediated cleavage occurs at duplex-single-strand junctions. Moreover, cleavage occurs only on the strand containing the 3' single-stranded tail. Thus, both biochemical and genetic evidence indicate a role for the Rad1-Rad10 complex in the cleavage of specific recombination intermediates. Furthermore, these data suggest that Rad1-Rad10 endonuclease incises DNA 5' to damaged bases during nucleotide excision repair.

Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair.

DNA ligase I belongs to a family of proteins that bind to proliferating cell nuclear antigen (PCNA) via a conserved 8-amino-acid motif [1]. Here we examine the biological significance of this interaction. Inactivation of the PCNA-binding site of DNA ligase I had no effect on its catalytic activity or its interaction with DNA polymerase beta. In contrast, the loss of PCNA binding severely compromised the ability of DNA ligase I to join Okazaki fragments. Thus, the interaction between PCNA and DNA ligase I is not only critical for the subnuclear targeting of the ligase, but also for coordination of the molecular transactions that occur during lagging-strand synthesis. A functional PCNA-binding site was also required for the ligase to complement hypersensitivity of the DNA ligase I mutant cell line 46BR.1G1 to monofunctional alkylating agents, indicating that a cytotoxic lesion is repaired by a PCNA-dependent DNA repair pathway. Extracts from 46BR.1G1 cells were defective in long-patch, but not short-patch, base-excision repair (BER). Our results show that the interaction between PCNA and DNA ligase I has a key role in long-patch BER and provide the first evidence for the biological significance of this repair mechanism.

The late, but not early, asthmatic response is dependent on IL-5 and correlates with eosinophil infiltration.

Early-phase reactions (EPRs) and late-phase reactions (LPRs) are characteristic features of bronchial asthma, although the pathogenetic mechanisms responsible for each of the responses are not fully defined. A murine model of EPRs and LPRs was developed to investigate the role of IL-5 and eosinophils in development of both responses. After initial intraperitoneal sensitization and airway challenge to ovalbumin (OVA), mice were provoked by additional exposure to OVA. An EPR, characterized by a transient increase in airway responsiveness, was observed 5-30 minutes after antigen provocation. This response was followed by an LPR that reached its maximum at 6 hours after challenge and was characterized by increased airway responsiveness and significant lung eosinophilia. The EPR was blocked by cromoglycate and albuterol, whereas the LPR was abolished by cromoglycate and hydrocortisone. Before provocation with allergen, administration of anti-IL-5 antibody prevented the influx of eosinophils into the lung tissue and abolished the LPR but not EPR. These results suggest that IL-5 and eosinophils are essential for development of the LPR, but not EPR, in this model.

An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase.

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.

Morbidity in patients waiting for tonsillectomy in Cardiff: a cross-sectional study.

OBJECTIVE: Our aim was to determine the morbidity of patients awaiting tonsillectomy. DESIGN: The study comprised a questionnaire survey of 379 children and 278 adults waiting over 12 months for tonsillectomy. OUTCOME MEASURES: These comprised frequency of infection, sickness absence and continuing desire for surgery. RESULTS: Response rates were 70 per cent (children) and 60 per cent (adults). Morbidity was similar in adults and children, and in those waiting more or less than two years. In the six months prior to the study, 86 per cent of children and 83 per cent of adults had had tonsillitis. Sixty per cent of children and 50 per cent of adults had had three or more episodes. Sixty-two per cent of children and 59 per cent of adults had had at least one long episode of tonsillitis, and 29 per cent of children and 24 per cent of adults had had more than three long episodes. Eighty-nine per cent of children had missed school at least once, compared with 71 per cent of adults missing work at least once (p = 0.01). The frequency of infection was significantly associated with patients' desire for surgery (p < 0.001). CONCLUSIONS: Patients awaiting tonsillectomy experience considerable morbidity. This study does not support the hypothesis that untreated patients will 'outgrow' their condition.

Completion of base excision repair by mammalian DNA ligases.

Three mammalian genes encoding DNA ligases--LIG1, LIG3, and LIG4--have been identified. Genetic, biochemical, and cell biology studies indicate that the products of each of these genes play a unique role in mammalian DNA metabolism. Interestingly, cell lines deficient in either DNA ligase I (46BR.1G1) or DNA ligase III (EM9) are sensitive to simple alkylating agents. One interpretation of these observations is that DNA ligases I and III participate in functionally distinct base excision repair (BER) subpathways. In support of this idea, extracts from both DNA ligase-deficient cell lines are defective in catalyzing BER in vitro and both DNA ligases interact with other BER proteins. DNA ligase I interacts directly with proliferating cell nuclear antigen (PCNA) and DNA polymerase beta (Pol beta), linking this enzyme with both short-patch and long-patch BER. In somatic cells, DNA ligase III alpha forms a stable complex with the DNA repair protein Xrcc1. Although Xrcc1 has no catalytic activity, it also interacts with Pol beta and poly(ADP-ribose) polymerase (PARP), linking DNA ligase III alpha with BER and single-strand break repair, respectively. Biochemical studies suggest that the majority of short-patch base excision repair events are completed by the DNA ligase III alpha/Xrcc1 complex. Although there is compelling evidence for the participation of PARP in the repair of DNA single-strand breaks, the role of PARP in BER has not been established.

DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis.

Several newly identified tumor suppressor genes including ATM, NBS1, BRCA1 and BRCA2 are involved in DNA double-strand break repair (DSBR) and DNA damage-induced checkpoint activation. Many of the gene products involved in checkpoint control and DSBR have been studied in great detail in yeast. In addition to evolutionarily conserved proteins such as Chk1 and Chk2, studies in mammalian cells have identified novel proteins such as p53 in executing checkpoint control. DSBR proteins including Mre11, Rad50, Rad51, Rad54, and Ku are present in yeast and in mammals. Many of the tumor suppressor gene products interact with these repair proteins as well as checkpoint regulators, thus providing a biochemical explanation for the pleiotropic phenotypes of mutant cells. This review focuses on the proteins mediating G1/S, S, and G2/M checkpoint control in mammalian cells. In addition, mammalian DSBR proteins and their activities are discussed. An intricate network among DNA damage signal transducers, cell cycle regulators and the DSBR pathways is illustrated. Mouse knockout models for genes involved in these processes have provided valuable insights into their function, establishing genomic instability as a major contributing factor in tumorigenesis.

The role of estrogen in the control of rat osteocyte apoptosis.

We have previously shown that estrogen withdrawal by gonadotrophin-releasing hormone analogs (GnRHa) induces osteocyte death via apoptosis in human bone. Although it is likely that the increase in osteocyte death via apoptosis was related to the loss of estrogen, these experiments could not rule out a direct role for the GnRHa. Therefore, in this study, we have used a rat model of ovariectomy (OVX) to determine whether the effect of estrogen withdrawal extends to other species and to clarify the role of estrogen in the maintenance of osteocyte viability. Twelve 9-week-old rats were divided into three treatment groups: sham operated (SHAM) (n = 4), OVX (n = 4), and OVX + estrogen (E2) (25 microg/day) (n = 4). At 3 weeks following the start of treatment, tibial bones were removed. The percentage of osteocytes displaying DNA breaks, using an in situ nick-translation method, was significantly higher in the OVX group compared with the SHAM control in both cortical bone (10.04% vs. 2.31%, respectively; p < 0.0001) and trabecular bone (6.44% vs. 1.58%, respectively; p = 0.003). Addition of estrogen in the OVX animals completely abrogated the increase in osteocyte apoptosis in cortical bone (0.78%) and trabecular bone (1.17%). The percentage of apoptotic osteocytes decreased with increasing distance from the primary/secondary spongiosa interface below the growth plate in the OVX model and the OVX + E2 model. Nuclear morphology and electrophoresis of DNA confirmed the presence of apoptotic cells in the samples. In conclusion, OVX in the rat results in an increase in osteocyte apoptosis as a direct or indirect result of E2 loss. Addition of estrogen in the OVX animals prevents this increase in osteocyte apoptosis. These data confirm an important role for estrogen in the control of osteocyte apoptosis and the maintenance of osteocyte viability. Estrogen deficiency might, through compromising the viability of osteocyte networks, reduce the ability of bone to respond appropriately to loading.

Preferential binding of Staphylococcus aureus to skin sites of Th2-mediated inflammation in a murine model.

Staphylococcus aureus is found on over 90% of atopic dermatitis skin lesions and is thought to contribute to skin inflammation via the production of potent exotoxins. In contrast, less than 5% of normal subjects harbor S. aureus. This suggests that an atopic immune response itself may play a role in preferential binding of S. aureus to the skin. To examine this issue more directly, we analyzed the S. aureus binding characteristics of skin in mice undergoing different T helper type 1 cell versus T helper type 2 cell inflammatory responses using a novel in vitro bacterial binding assay. BALB/C female mice were first sensitized to ovalbumin with alum or ovalbumin with complete Freund's adjuvant to induce T helper type 2 or T helper type 1 responses, respectively. Mice were then challenged intradermally with either saline (control) or ovalbumin. Forty-eight hours later, skin specimens were obtained from the challenge sites, and the number of S. aureus binding to each skin section was quantitated. Bacterial binding was found to be significantly greater at skin sites of BALB/C mice that had been ovalbumin/alum sensitized compared with ovalbumin/complete Freund's adjuvant sensitized (p < or = 0.01). When compared to the ovalbumin sensitized/challenged skin of wild type BALB/C mice or interferon-gamma gene knockout mice, interleukin-4, but not interferon-gamma, gene knockout mice had significantly less S. aureus binding at their ovalbumin sensitized/challenged skin sites. Mutant S. aureus strains that lacked either fibronectin- or fibrinogen-binding protein expression showed significantly reduced S. aureus binding compared with the parent wild type strain (p < 0.005). Moreover, preincubation of the wild type bacteria with fibronectin or fibrinogen, but not collagen, resulted in significantly less skin binding of S. aureus (p < 0.01). Incubation of skin with interleukin-4, and less so with interferon-gamma, led to more binding of wild type S. aureus but not of an S. aureus mutant deficient in fibronectin binding protein expression. After interleukin-4 incubation, but not interferon-gamma, epidermal immunoreactivity for fibronectin was observed in murine skin explants. These results show that a T helper type 2 inflammatory environment can promote skin binding by S. aureus and that this binding is mediated by fibronectin and fibrinogen.

The death of osteocytes via apoptosis accompanies estrogen withdrawal in human bone.

Estrogen withdrawal in women leads initially to rapid bone loss caused by increased numbers or activity of osteoclasts. We previously have noted apoptosis of lacunar osteocytes associated with conditions of high bone turnover. Therefore, in this study, we investigated whether the increased bone loss associated with GnRH analogue (GnRH-a)-induced estrogen withdrawal affects osteocyte viability in situ in a way that would be directly contrary to the effect of estrogens on osteoclast viability. Transiliac biopsies were obtained from six premenopausal women, between 30-45 yr old, diagnosed as having endometriosis. Biopsies were taken before and after 24 weeks of GnRH-a therapy. Biopsies were snap-frozen and cryostat sectioned. Osteocyte viability, determined by the presence of lactate dehydrogenase (LDH) activity, was reduced in all but one subject after treatment. Furthermore, in every subject, the proportion of osteocytes showing evidence of DNA fragmentation typical of apoptosis increased, as demonstrated using in situ DNA nick translation (P = 0.008). Gel electrophoresis of extracted DNA and morphological studies of chromatin condensation and nuclear fragmentation confirmed that changes typical of apoptosis were affecting the osteocytes. It was concluded that GnRH-a therapy caused a higher prevalence of dead osteocytes in iliac bone, probably caused by the increase in the observed proportion of osteocytes showing apoptotic changes. The capacity of bone to repair microdamage and to modulate the effects of mechanical strain is currently believed to be dependent on osteocyte viability. Our findings have therefore revealed a possible mechanism whereby estrogen deficiency could lead to increased bone fragility with or without an accompanying net bone loss.

The effect of isoenzyme-selective PDE inhibitors on methacholine-induced contraction of guinea-pig and rat ileum.

1. We have examined the effects of the isoenzyme-selective phosphodiesterase (PDE) inhibitors, vinpocetine (type 1), siguazodan (type 3), rolipram (type 4) and zaprinast (type 5) and the non-selective PDE inhibitor enprofylline on methacholine (MCh) contractile concentration-response curves on guinea-pig and rat isolated ileum. 2. In guinea-pig ileum, vinpocetine (10-300 microM), zaprinast (1-300 microM) and enprofylline (100-1000 microM) produced a concentration-dependent depression of the maximum response (Emax) to MCh only without effect on the MCh EC50 values (rank order of potency: zaprinast > vinpocetine > enprofylline). In contrast, siguazodan (10-300 microM) and rolipram (10-300 microM) produced a rightward displacement of the MCh concentration-response curve (increase in EC50: rank order; rolipram > siguazodan), with effects on the MCh maximum seen only at higher concentrations. 3. In the rat ileum, vinpocetine (10-300 microM), zaprinast (0.1-300 microM) and enprofylline (100-1000 microM) caused depression of the MCh maximum contraction (rank order: zaprinast > vinpocetine > enprofylline). Low concentrations of rolipram and siguazodan had no significant effect on the MCh maximum. In the presence of higher concentrations (> 100 microM) of rolipram and siguazodan, a maximum response was not achieved at the highest concentration of MCh tested. As in the guinea-pig ileum, only rolipram (10-300 microM) and siguazodan (10-300 microM) produced a significant, concentration-dependent, rightward displacement of the MCh concentration-response curve (increase in EC50: rank order: rolipram > siguazodan). 4. In the guinea-pig ileum, isoprenaline (0.1 microM) produced a rightward displacement (approximately 3 fold) of the MCh concentration-response curve, accompanied by a significant depression of the maximum response. Increasing the isoprenaline concentration (1 microM) had no further effect on either parameter. Sodium nitroprusside (SNP, > or = 10 microM) produced a concentration-dependent depression of the MCh maximum without an effect on the EC50. 5. In the rat ileum, isoprenaline (1 microM) produced a concentration-dependent rightward displacement (approximately 2.8 fold) of the MCh concentration-response curve with depression of the MCh maximum at higher (> or = 100 microM) concentrations. SNP produced depression of the MCh maximum at a concentration of 10 microM and above. Effects on the MCh EC50 were seen only at 100 and 300 microM. 6. In guinea-pig ileum, isoprenaline (0.1 microM) in combination with rolipram (10 microM) further increased the MCh EC50 and reduced the MCh maximum. The combination of SNP (10 microM) with zaprinast (0.1 microM) produced no further significant effect than SNP alone. 7. In rat ileum, isoprenaline (1 microM) in combination with rolipram (10 microM) further increased the EC50 and reduced the maximum. SNP (10 microM) had no significant effect on either the MCh maximum or EC50. A combination with zaprinast (1 microM) had no further effect. 8. In conclusion, all the PDE inhibitors tested produced a concentration-dependent inhibition of the MCh concentration-response curve, indicating a modulator role for the PDE isoenzymes in gastrointestinal smooth muscle contractility. The PDE inhibitors that elevate cyclic GMP produced a depression of the MCh maximum response only, whilst those that elevate cyclic AMP produced a rightward displacement of the MCh concentration-response curve. This was confirmed by the use of isoprenaline and SNP. This difference in the type of inhibition produced by these PDE isoenzyme inhibitors may reflect a different intracellular site/mechanism by which the cyclic AMP- and cyclic GMP-activated kinases act functionally to antagonize the contractile response.

Comparison of the effects of selective inhibitors of phosphodiesterase types III and IV in airway smooth muscle with differing beta-adrenoceptor subtypes.

1. The relaxant properties of the type IV adenosine 3',5'-cyclic monophosphate phosphodiesterase (cyclic AMP PDE) inhibitor, rolipram and the beta 2-selective and non-selective beta-adrenoceptor agonists salbutamol and isoprenaline, were compared on the guinea-pig, bovine, and mouse trachea and porcine bronchus all precontracted with methacholine (EC30). 2. Rolipram and both beta-agonists produced concentration-dependent reversal of the methacholine-induced tone in the four airway preparations. 3. Isoprenaline and salbutamol were similar in potency on the guinea-pig (-log10IC50:8.43, 8.06) and bovine (-log10 IC50:8.52, 8.40) airways. In contrast, salbutamol was much less potent than isoprenaline on the mouse trachea (> 1000 fold) and the porcine bronchus (> 100,000 fold). 4. The potency of rolipram approached that of isoprenaline on the guinea-pig and bovine trachea (beta 2-adrenoceptors predominate). However, rolipram was significantly less active than isoprenaline on the porcine bronchus (1000 fold) and mouse trachea (> 2000 fold) where beta 2-adrenoceptors predominate. 5. Siguazodan, the type III cyclic AMP PDE inhibitor, produced concentration-dependent relaxations of the porcine bronchus and guinea-pig trachea contracted with methacholine. Siguazodan was 100 fold more active than rolipram in pig tissues indicating the type III isoenzyme may be of greater functional significance in this tissue. In contrast, siguazodan was 15 times less potent that rolipram in guinea-pig airways suggesting a greater role for the type IV PDE. 6. These findings may reflect a possible relationship between the beta 2-adrenoceptor subtype and the functional importance of the type IV PDE isoenzyme. A similar relationship may exist between beta 1-adrenoceptors and the PDE type III isoenzyme.