Mårten Sandberg

Abstract: BACKGROUND: Reduced transport time of patients from the scene of an accident to definitive surgical treatment is one important reason to employ ambulance helicopters on trauma missions. However, if the helicopter is unable to land close to the scene, the transport time may be increased compared to transport with ground ambulance, due to time-consuming transfer of the patient between vehicles. OBJECTIVE: The objective of this study was to evaluate how the landing site, as determined by distance from the scene, and rapid sequence intubation (RSI) affected on-scene time (OST). METHODS: This was a prospective observational study performed during a 12-month period in a mixed urban and rural anesthesiologist-staffed Helicopter Emergency Medical Service in Norway. Data regarding the landing sites, the accident, and patient treatment were recorded. RESULTS: A total of 252 primary trauma missions were included in the study. In 75% of the missions, the aircraft landed<50 meters from the scene, and in 7% the distance exceeded 200 meters. Mean OST when the patient was not intubated was 14.5min (median 14min). When an RSI was performed, the mean OST was significantly higher (22.7min, median 20min; p<0.001). CONCLUSION: Usually, a helicopter can land close to the accident scene and the location of the landing site does not contribute to a delay in arrival of the patient at the hospital. The OST is significantly higher, however, in those patients who receive endotracheal intubation before take-off. This reflects the time needed for intubation, as well as the increased complexity and workload when the patient is severely injured.

Abstract: BACKGROUND: Hypoxemia may occur during rapid sequence intubation (RSI). This study establishes the incidence of this adverse event in patients intubated by physicians in a helicopter emergency service in Norway. METHODS: This was a prospective, observational study of all RSIs performed by helicopter emergency service physicians during a 12-month period. Hypoxemia was defined as a decrease in Spo(2) values to below 90% or a decrease of more than 10% if the initial Spo(2) was less than 90%. RESULTS: A total of 122 prehospital intubations were performed during the study period. Spo(2) data were available for 101 (82.8%) patients. Hypoxemia was present in 11 (10.9%) patients. CONCLUSIONS: Prehospital, RSI-related hypoxemia rates in this study are lower than reported rates in similar studies and are comparable with in-hospital rates. Prehospital RSI may accordingly be considered a safe procedure when performed by experienced physicians with appropriate field training.

Abstract: BACKGROUND: Airway management of entrapped patients is challenging and alternatives to endotracheal intubation with a Macintosh laryngoscope must be considered. In this study, the GlideScope Ranger video laryngoscope has been evaluated as an alternative to standard laryngoscopy. METHODS: Eight anaesthesiologists from a Helicopter Emergency Medical Service intubated the trachea of a Laerdal SimMan manikin using the studied laryngoscopes in two scenarios: (A) unrestricted access to the manikin in an ambulance and (B) no access from the head end, simulating an entrapped patient. The time used to secure the airway and the scored level of difficulty were the main variables. RESULTS: In scenario A, all anaesthesiologists managed to secure the airway using both techniques within the 60-s time limit. In scenario B, all secured the airway when using the video laryngoscope, while 50% succeeded with endotracheal intubation using the Macintosh laryngoscope. The difference in the success rate was statistically significant (P=0.025). There were no significant differences in the time spent on endotracheal intubation in the two scenarios or between the devices. All stated that the availability of a video laryngoscope would make drug-facilitated intubation a realistic alternative when access to patients is limited. The lack of visual control when using the Macintosh laryngoscope excludes this technique in real-life settings. CONCLUSION: This study suggests that the GlideScope Ranger may be merited in situations requiring endotracheal intubation by an experienced intubator in patient entrapment. Further studies are required to clarify whether performance in patients mimics that in a manikin.

Abstract: BACKGROUND: Medical emergency motorcycles (MEM) can be used in time-critical conditions like cardiac arrest and multi-traumatized patients in an attempt to reduce the response time. Other potential benefits with MEM are more efficient patient evaluation, reduction of unnecessary EMS car ambulance missions and reduced cost. The potential benefits have been evaluated in this study. The incidence of accidents when operating the vehicle was also of interest. METHODS: A prospective study was performed when MEM was introduced as a trial in an urban ambulance service in Norway. RESULTS: A total of 703 MEM missions were registered in the period. The mean emergency driving time was significantly shorter for the MEM than for the ambulance car located at the same station (6 min 24 seconds vs. 6 min 54 seconds). In addition to time-critical conditions, the MEM was used to evaluate patients when the need for emergency medical assistance was uncertain, and this practice lead to a reduced number of unnecessary car ambulance missions. No accidents involving the MEM were registered in the study period. The hourly cost of running the MEM was 29 euro vs. 75 euro for a car ambulance. However, the actual cost benefit is smaller since the weather conditions make it impossible to run a MEM in wintertime. CONCLUSION: The small reduction in driving time when using a MEM instead of a car ambulance was statistically significant but probably of little clinical importance. The number of unnecessary car ambulance missions was reduced. It was cheaper to operate a MEM than a car ambulance, but the cost-effectiveness was reduced since the MEM could not operate 12 months a year. The lack of accidents may be contributed to the extensive training of the drivers and the fact that the vehicle was operated in daylight only.

Abstract: This article is intended as a generic guide to evidence-based airway management for all categories of pre-hospital personnel. It is based on a review of relevant literature but the majority of the studies have not been performed under realistic, pre-hospital conditions and the recommendations are therefore based on a low level of evidence (D). The advice given depends on the qualifications of the personnel available in a given emergency medical service (EMS). Anaesthetic training and routine in anaesthesia and neuromuscular blockade is necessary for the use of most techniques in the treatment of patients with airway reflexes. For anaesthesiologists, the Task Force commissioned by the Scandinavian Society of Anaesthesia and Intensive Care Medicine recommends endotracheal intubation (ETI) following rapid sequence induction when securing the pre-hospital airway, although repeated unsuccessful intubation attempts should be avoided independent of formal qualifications. Other physicians, as well as paramedics and other EMS personnel, are recommended the lateral trauma recovery position as a basic intervention combined with assisted mask-ventilation in trauma patients. When performing advanced cardiopulmonary resuscitation, we recommend that non-anaesthesiologists primarily use a supraglottic airway device. A supraglottic device such as the laryngeal tube or the intubation laryngeal mask should also be available as a backup device for anaesthesiologists in failed ETI.

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Abstract: A 50-year-old patient had status epilepticus and no adequate reactions nine days after prolonged out-of-hospital cardiac arrest. The cause of the arrest was acute myocardial infarction which was treated successfully with percutaneous cardiac intervention (PCI) and a stent placement. He was treated with therapeutic hypothermia (33 degrees C) for 24h and in intensive care with respiratory support for 42 days. One year later he has fully recovered and is back to normal life and academic work. The previously reported 100% prognosis of a poor neurological outcome in the presence of seizures 72 h post arrest may need to be re-examined after introduction of therapeutic hypothermia.

Abstract: BACKGROUND: The purpose of this study was to describe the use of a rapid response car (RRC) as a supplement to the ambulance helicopter in a mixed urban/rural region in Norway. METHODS: Data from all the requested missions were collected from standard flight records. Operational factors, patient characteristics, primary diagnosis, treatment and modes of transport were registered and analyzed retrospectively. RESULTS: In 1999-2001, a total of 4777 requests were included in the study, resulting in the initiation of 3172 helicopter and 752 RRC missions. In the RRC missions, 224 patients received advanced medical treatment that would otherwise not have been provided. For 181 patients, the availability of the RRC was crucial for receiving the treatment of the helicopter emergency medical services (HEMS). The cost of equipping the base with the RRC increased the annual budget by less than one percent. CONCLUSION: The RRC was essential for solving missions in periods of non-flying conditions. The RRC increased the availability of the advanced prehospital life support offered by the HEMS in this region. Taking the modest increase in cost into consideration, it seems reasonable that this HEMS, covering mixed urban and rural areas, is equipped with such a vehicle.

Abstract: Phosphodiesterases are enzymes that catalyze the degradation of the cyclic nucleotides, cyclic AMP and cyclic GMP, to the corresponding 5' nucleotide monophosphates. Ten different phosphodiesterase families have been described to date. These enzymes exist as homodimers and there is structural similarity between the different families. However, they differ in several respects like selectivity for cyclic nucleotides, sensitivity for inhibitors and activators, physiological roles and tissue distribution. Interest in these enzymes has increased tremendously, both within the medical community and in the general public as a consequence of sildenafil (Viagra), the medication recently introduced for the treatment of erectile dysfunction. Sildenafil mediates its effects by inhibiting phosphodiesterase 5. Some biochemical and molecular biological aspects of this enzyme are presented here. To achieve satisfactory erection, normal penile innervation is required. Nitrogen monoxide, the transmitter substance in these nerves, activates guanylyl cyclase, thereby increasing cyclic GMP production. The increased levels of cyclic GMP cause relaxation of smooth muscles in penile vessels and this leads to an erection. Erection is dependent on elevated levels of cyclic GMP and sildenafil mediates its effects by inhibiting the degradation of cyclic GMP. Other functions that are mediated by the phosphodiesterases explain visual disturbances, flushing and decreased blood pressure that are some of the side effects seen with sildenafil treatment. Furthermore, the potentially fatal consequence of combining sildenafil and nitrovasodilators is discussed.

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Abstract: The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.

Abstract: A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein-coupled cell-surface receptors have their signals converge at one sole second messenger, cAMP. The question of how specificity can be maintained in a signal-transduction system in which many extracellular signals leading to a vast array of intracellular responses are all mediated through one second-messenger system has been the subject of thorough investigation and a great deal of speculation. An increasing number of cAK isozymes, consisting of homo- or heterodimers of R subunits (RIalpha, RIbeta, RIIalpha, RIIbeta) with associated catalytic subunits (C alpha, Cbeta, Cgamma), may, at least in part, explain this specificity. The various cAK isozymes display distinct biochemical properties, and the heterogeneous subunits of cAK reveal cell-specific expression and differential regulation at the level of gene transcription, mRNA stability, and protein stability in response to a wide range of hormones and other signaling substances. The existence of a number of anchoring proteins specific to either RIIalpha or RIIbeta, and which localize cAKII isozymes toward distinct substrates at defined subcellular loci, strongly supports the idea that specific functions can be assigned to the various cAK isozymes. The demonstration that selective activation of cAKI is necessary and sufficient for cAMP-mediated inhibition of T-cell proliferation, and the observation that T-cell activation is associated with redistribution and colocalization of cAKI to the TCR, is also compatible with the notion of isozyme-specific effects.

Abstract: The type I cGMP-dependent protein kinase (cGK) has been shown to play a crucial role in the relaxation of vascular smooth muscle by lowering the intracellular level of calcium. Two isoforms of type I cGK have been described, type I alpha and type I beta, differing only in their N-terminal parts. This report describes the cloning of the gene PRKG1 encoding both human type I cGK isoforms. PRKG1 is a single-copy gene consisting of 19 exons encompassing at least 220 kb. Several of the splice sites previously observed in the Drosophila melanogaster DG2 gene have been conserved in PRKG1, and these conserved splice sites correlated well with the boundaries between several of the previously proposed functional domains of type I cGK. The first two exons of the type I cGK gene were shown to encode the type I alpha- and type I beta-specific parts of the cGK. Using 5'-rapid amplification of cDNA ends, potential sites for transcription initiation were identified 5' upstream of both these exons. Northern blot analyses demonstrated distinct patterns of expression of the isoforms of type I alpha and I beta cGK in different human tissues.

Abstract: Both cyclic GMP-dependent protein kinase (cGK) and cyclic AMP-dependent protein kinase (cAK) contain two distinct cyclic nucleotide-binding sites referred to as fast and slow sites based on cyclic nucleotide dissociation behavior. In cAK, the fast site lies amino-terminal to the slow site, and sequence homologies between cAK and cGK have suggested similar positioning for the sites in cGK. Recombinant human type Ibeta cGK (wild type (WT) cGK) was overexpressed, and the properties of purified WT cGK and native type Ibeta cGK were similar. cGK was mutated singly at Thr-193 (T193A, T193V, and T193S) and Thr-317 (T317A, T317V, and T317S), which have been predicted to provide cGMP specificity in the cGMP-binding sites of cGK; a double mutant (T193A/T317A) was produced also. Compared with WT cGK, half-maximal activation (Ka) of mutant cGKs by cGMP was increased 2- (T317A), 27- (T193A), or 63-fold (T193A/T317A), but the Ka for cAMP of these mutants was essentially unchanged. The T193A and T193V mutants had a large increase in the rate of the slow component of [3H]cGMP dissociation, but in the T317A and T317V mutants, there was no change in the slow component. The T193S and T317S mutants had only minor effects on [3H]cGMP dissociation, thus establishing the importance of the hydroxyl group of Thr-193 and -317 for cGMP binding to cGK. Thus, in type Ibeta cGK, the slow cGMP-binding site is identified as the amino-terminal site in contrast to the order assigned to the fast and slow cAMP-binding sites of cAK.

Abstract: The type II cGMP-dependent protein kinase is an enzyme originally isolated from the small intestine, and is thought to be involved in the regulation of intestinal ion transport and fluid secretion. A complementary DNA clone encoding a part of the human type II cGMP-dependent protein kinase was isolated from a cerebellum library. Based on sequence information from this complementary DNA, the 5'-end of the type II cGMP-dependent protein kinase was amplified from human brain messenger RNA using polymerase chain reaction. The composite complementary DNA encoded a 762 amino acid protein with a calculated molecular mass of 87.4 kDa. Messenger RNAs encoding the type II cGMP-dependent protein kinase were detected in small intestine, colon and prostate. By using polymerase chain reaction and Southern blotting on somatic cell hybrids, the gene encoding, the type II cGMP-dependent protein kinase was mapped to human chromosome 4q13.1-q21.1.

Abstract: The role of the cGMP-dependent protein kinase (cGK) and one of its major substrates, the vasodilator-stimulated phosphoprotein (VASP), in the regulation of a receptor-evoked calcium response was investigated. The human type I beta cGK was stably transfected in human embryonic kidney 293 cells and Swiss mouse 3T6 fibroblasts, which contained significant or no detectable levels of the focal adhesion protein VASP, respectively. Western blot analysis and protein kinase activity measurements demonstrated an 8-fold overexpression of cGK-I beta in 293 cells (7-fold in 3T6 cells), representing an intracellular cGK concentration of 0.33 microM. In experiments with intact 293 cells expressing cGK-I beta, beta-phenyl-1,N2-etheno-cGMP and 8-(p-chlorophenylthio)-cGMP were capable of converting up to 30-40% of the 46-kDa VASP to its 50-kDa phospho- form, equivalent to results observed with cGMP analogs that cause a marked inhibition of the stimulated Ca2+ transient in intact human platelets. In contrast to platelets, preincubation of fura-2-loaded 293 and 3T6 cells with 8-(p-chlorophenylthio)-cGMP did not significantly inhibit thrombin-evoked calcium transients, although sufficient cGK-mediated VASP phosphorylation was clearly detectable under these conditions in cGK-I beta-expressing 293 cells. These results demonstrate that cGK inhibition of agonist-evoked calcium mobilization is not a mechanism common to all cell types and that VASP phosphorylation may not be an essential or sufficient component of the cGK effect on calcium levels. In contrast, the observed VASP phosphorylation mediated by recombinant human cGK-I beta in intact 293 cells does support the hypothesis that focal adhesions and their associated proteins are important cellular sites of cGK action.

Abstract: Using a human cDNA (complementary DNA) encoding the regulatory subunit RI alpha of cAMP-dependent protein kinases (PKA) as a probe, a pseudogene for this PKA isoform was isolated from a human genomic library. The human RI alpha pseudogene was 89% similar to the open reading frame of the expressed human RI alpha at the nucleotide level. Several stop codons were found, indicating that the pseudogene does not encode a functional protein. The pseudogene also contained several frameshift mutations, small deletions, and insertions. No introns were found in the region corresponding to the open reading frame of the expressed RI alpha cDNA. Specific oligonucleotides for the RI alpha gene and pseudogene were constructed and used as primers in polymerase chain reaction (PCR) to amplify human DNA from healthy blood donors. Probing of the PCR products using oligonucleotides specific for the RI alpha gene and RI alpha pseudogene, respectively, showed the presence of both genes in the human genome. When DNA extracted from various somatic cell hybrids was amplified, it was shown that the RI alpha gene was located on human chromosome 17, whereas the RI alpha pseudogene was located in the p21-p31 region on chromosome 1. This is the first report describing a pseudogene for a subunit of cAMP-dependent protein kinases from any species.

Abstract: We have recently characterized cDNAs and genomic DNA fragments for human type I cGMP-dependent protein kinase (cGK). By probing human x hamster hybrid cell lines with a 1.2-kb intron fragment from the human type I cGK gene, we identified a 5.9-kb BglII restriction fragment and localized it to human chromosome 10. In situ hybridization analyses using 3H-labeled cDNA and genomic DNA probes for the human type I cGK to human metaphase chromosomes supported the somatic cell hybrid data and indicated that the gene (PRKG1B; protein kinase, cGMP-dependent) maps to 10p11.2----q11.2.

Abstract: A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3',5'-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.

Abstract: Using a cDNA probe for the type I alpha regulatory subunit, two mRNA species (1.5 and 3.0 kb in length) were detected in human testis. From a human testis cDNA-library a 3.0 kb clone, containing the entire reading frame of the protein, was isolated. Comparison of the nucleotide sequence of this clone to the sequence of a 1.5 kb cDNA clone earlier reported, showed that the longer clone was identical to the shorter but extended another 1.5 kb in the 3' end. Sequencing data together with Northern blot analysis indicated that the two mRNA species for human type I alpha regulatory subunit were generated from the same gene by the use of different polyadenylation site signals.

Abstract: In this study we report the isolation and characterization of three overlapping cDNA clones for the type I beta isozyme of cGMP-dependent protein kinase (cGK) from human placenta libraries. The composite sequence was 3740 nucleotides long and contained 58 nucleotides from the 5'-noncoding region, an open reading frame of 2061 bases including the stop codon, and a 3'-noncoding region of 1621 nucleotides. The predicted full-length human type I beta cGK protein contained 686 amino acids including the initiator methionine, and had an estimated molecular mass of 77,803 Da. On comparison to the published amino acid sequence of bovine lung I alpha, human placenta I beta cGK differed by only two amino acids in the carboxyl-terminal region (amino acids 105-686). In contrast, the amino-terminal region of the two proteins was markedly different (only 36% similarity), and human I beta cGK was 16 amino acids longer. In a specific region in the amino-terminus (amino acids 63-75), 12 out of 13 amino acids of the human I beta cGK were identical to the partial amino acid sequence recently published for a new I beta isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human I beta cGK mRNA, 7 kb in size, in human uterus and weakly in placenta. An mRNA of 7 kb was also observed in rat cerebellum, cerebrum, lung, kidney, and adrenal, whereas an mRNA doublet of 7.5 and 6.5 kb were observed in rat heart. Comparison of Northern and Western blot analyses demonstrated that the mRNA and protein for cerebellar cGK increased during the development of rats from 5 to 30 days old, whereas the 6.5 kb mRNA in rat heart declined.

Abstract: The regulatory subunit of cAMP-dependent protein kinase designated RII beta (RII51) has previously been shown to be the product of a separate gene. This was accomplished by the molecular cloning of a partial cDNA clone estimated to lack 30-45 nucleotides of the 5' end of the coding region. We hereby report the isolation of a cDNA clone for RII beta from rat granulosa cells, extending 43 nucleotides further 5' compared with the previously published cDNA sequence, and from which the entire amino acid sequence (415 residues) of the rat RII beta protein can be deduced. A cAMP regulated mRNA of 3.2 kilobases (kb) for RII beta was detected by the isolated cDNA in rat Sertoli cells.

Abstract: In recent years a multiplicity in isoforms of cAMP-dependent protein kinases has been revealed. Gene products for four different regulatory subunits (RI alpha, RI beta, RII alpha, RII beta) and two different catalytic subunits (C alpha, C beta) have been identified. We hereby present the molecular cloning of rat cDNAs for RII beta, as well as full-length human cDNAs for RII beta and RI alpha. The amino acid sequences deduced from the cDNAs of the regulatory subunits, revealed dissimilarities which were primarily confined to the N-terminal part of the protein. Based on the vital role in testicular function played by gonadotropin-induced activation of cAMP-dependent protein kinases, mRNA levels for the various subunits of cAMP-dependent protein kinase have been studied in rat testis. A clear pattern of cellular localization of mRNAs for the various subunits of cAMP-dependent protein kinase has been demonstrated. Furthermore, stimulation of Sertoli cells by FSH and cAMP elicited a differential response in mRNA levels for various subunits. A dramatic increase (30-40 fold) in the mRNA for RII beta (3.2 kb) was seen with cAMP stimulation, whereas such treatment had minor effects on mRNAs for RI alpha, RII alpha and C alpha. A distinct pattern of expression for various subunits of cAMP-dependent protein kinase was observed during germ cell differentiation. RI alpha and RI beta were expressed at high levels at early stages of spermatogenesis, whereas unique mRNAs for RII alpha and RII beta appeared in post-meiotic germ cells. Altogether, the present results demonstrate specific expression of mRNAs for different subunits of cAMP-dependent protein kinase in different cell types, during hormonal stimulation and during cellular differentiation. This indicates that the individual subunits may confer specific functional properties to the cAMP-dependent protein kinase holoenzyme and to the cAMP signal pathway of the cell.

Abstract: In the present study we have examined the effects of FSH, forskolin, and (Bu)2cAMP on messenger RNA (mRNA) levels for all known subunits of cAMP-dependent protein kinase in rat Sertoli cells, using newly developed complementary DNA (cDNA) probes. mRNAs for the three regulatory subunits [RI alpha, RII51, (RII beta), and RII54 (RII alpha)] and the catalytic subunit C alpha were shown to be present in cultured rat Sertoli cells, whereas mRNAs for the subunits designated RI beta and C beta were below the level of detection. A high-levelled, concentration-dependent increase in a 3.2 kilobase mRNA for RII51 was observed when cultured immature Sertoli cells were incubated with increasing concentrations of (Bu)2cAMP (10(-6) to 5 X 10(-3) M) for 16 h. Densitometric scanning indicated a maximal stimulation by (Bu)2cAMP of 30- to 40-fold. Incubation with forskolin (100 microM) and FSH (200 ng/ml) gave rise to a smaller but significant increase in mRNA for RII51. When cultured Sertoli cells were incubated in the presence of 10(-4) M (Bu)2cAMP for varying time periods, there was a biphasic regulation of mRNA for RII51. (Bu)2cAMP caused an initial increase in mRNA for RII51 with maximal levels obtained after 10-16 h, after which a time-dependent decrease was observed. For the other three subunits present in Sertoli cells (RI alpha, RII54, and C alpha) a smaller but significant stimulation by (Bu)2cAMP and forskolin (2-4 fold) was seen. The functional implications of these changes in mRNA levels for the different subunits of cAMP-dependent protein kinase have not yet been revealed. However, our data clearly demonstrate differential regulation of the various subunits of cAMP-dependent protein kinase in Sertoli cells. Furthermore, these results document the presence of distinct adaptational changes taking place at the level of cAMP-dependent protein kinase in response to long term elevation of cAMP.

Abstract: In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.

Abstract: Cystic fibrosis (CF) is a common autosomal recessive disease with significant morbidity and mortality. Defects in cAMP control mechanisms are implicated in the pathophysiology of the disease. The mutation causing CF has been localized to chromosome 7q22-7q31.1. We have used (1) somatic-cell hybrids containing this region of the human genome in a mouse background and (2) segregation analysis in families to exclude both the genes coding for a catalytic subunit and three distinct regulatory subunits of cAMP-dependent protein kinase as candidates for the gene defect in CF. Two of these genes--those for the human homologue of the mouse type I regulatory subunit and the human homologue of the rat type II regulatory subunit--map to human chromosome 7.

Abstract: Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes. The regulatory subunit (R) designated RII51 was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated C alpha, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the C alpha, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis. In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.

Abstract: A 1.5 kilobase (kb) cDNA clone containing the entire coding region for a regulatory subunit of type I cAMP-dependent protein kinase (RI) was isolated from a human testis cDNA library. The cDNA clone encodes a protein of 381 amino acids that shows 98% and 97% homology to the bovine skeletal muscle RI and rat brain RI, respectively. Northern blot analysis demonstrates two major mRNA-species (1.5 and 3.0 kb) in human testis and one mRNA-species (3.0 kb) in human T-lymphocytes.