Norepinephrine-induced interleukin-6 increase in rat hearts: differential signal transduction in myocytes and non-myocytes.

Continuous i.v. infusion of norepinephrine in rats has been shown to induce early interleukin (IL)-6 mRNA expression in the left ventricle (LV) which was followed by hypertrophy and fibrosis. In this study, two approaches were used. In the first, NE (0.1 mg/kg per hour) was infused i.v. in rats for several time periods, and freshly obtained ventricular myocardium was dissociated into myocyte (MC) and non-myocyte (NMC) fractions. Second, isolated adult MCs and fibroblasts were treated with NE (10 microM). NE infusion (4 h, in vivo) caused an 11-fold increase in IL-6 mRNA in both cell populations. In vitro treatment of isolated adult MCs for 2 h and of fibroblasts for 1 h with NE induced a 3.5- and 23-fold maximum increase, respectively, in IL-6 mRNA. After in vivo NE treatment, the expression of the mRNA of the transcriptional factor of IL-6, C/EBP-beta, was elevated earlier (after 45 min of NE infusion) than IL-6 mRNA (after 4 h) and was seen in MCs and NMCs. The mRNAs of both receptors of IL-6, the soluble IL6R and gp130, were increased subsequently to IL-6 mRNA. Gp130 was elevated after 24 h and, like IL6R, predominantly in NMCs. In contrast, the IL6R protein and the downstream regulator STAT3 were increased only in MCs after 24 h of NE infusion. The mRNA of C/EBP-delta, which is regulated by STAT3, was elevated only in myocytes.

Serum depletion induces cell loss of rat cardiac fibroblasts and increased expression of extracellular matrix proteins in surviving cells.

OBJECTIVE: Since reduced nutrient supply is one component of ischemia, we have studied the effect of serum depletion on the survival of fibroblasts isolated from adult rat hearts and on the expression and degradation of extracellular matrix (ECM) proteins. Furthermore, we measured the role of the cAMP-dependent pathway in these processes. METHODS: Isolated cardiac fibroblasts were grown to confluency in 10% serum containing medium. Serum was then removed and cell number was measured by use of a Coulter Counter. The activity of the cAMP response element binding protein (CREB) was investigated by Western blotting and subsequent use of the specific antibody which binds to the active form of the protein. The expression of colligin, collagen I and III, matrix metalloproteinases 2 (MMP-2), and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) was examined by ribonuclease protection assay (RPA) and Western blotting. Zymographic measurements were done to investigate gelatinase activity of MMP-2. RESULTS: Serum withdrawal caused the death of 36% of the cells during the first 8 h. CREB was strongly phosphorylated 5 min after serum removal. Activation persisted up to 8 h and decreased thereafter. The mRNA abundance of colligin, collagen I and III, MMP-2, and TIMP-2 started to increase after 5 and 10 h, respectively, reaching a maximum after 20-30 h and decreasing thereafter. Protein levels of collagen I, collagen III, colligin and TIMP-2 were higher after 24 h until up to 96 h. MMP-2 zymographic activity was elevated 15-fold after 72 h. Application of the protein kinase A (PKA) blocker RpcAMPS suppressed the increase in phosphorylation of CREB. The increase in collagen III and MMP-2 mRNA abundance and elevation of collagen I and III, and TIMP-2 protein was diminished by RpcAMPS. The rise of colligin protein was completely suppressed. The increase in MMP-2 zymographic activity was also attenuated. RpcAMPS improved survival rate from 56 to 84%. CONCLUSIONS: Serum depletion led to cell death of isolated cardiac fibroblasts. Survival was associated with the increase in the expression of various ECM proteins. The transcription factor CREB was activated after serum removal. Inhibition of PKA improved the serum depletion induced decrease in the survival rate. The increase in collagen I, collagen III, MMP-2, TIMP-2, and colligin evoked by serum depletion was also diminished by PKA inhibition.

Cardiac remodeling after long term norepinephrine treatment in rats.

OBJECTIVE: In this study we have tested the hypothesis that degradation of collagen by matrix metalloproteinase 2 (MMP-2) precedes the deposition of extracellular matrix (ECM) after long term norepinephrine (NE) treatment. METHODS: Female Sprague-Dawley rats received continuous i.v. infusion of NE (0.1 mg/kg.h) for 1, 2, 3, 4 and 14 days. Heart function and weight as well as expression of cardiac colligin and of collagen I and III were examined. Furthermore, we have assessed the degradation pathway of collagen by measuring the mRNA and activity of myocardial MMP-2 and tissue inhibitor of metalloproteinase 2 (TIMP-2) as well as the protein level of TIMP-2. RESULTS: NE induced hypertrophy predominantly of the left ventricle (LV) in a time-dependent manner. It increased the mRNAs of colligin, collagen I and III, and of MMP-2 and TIMP-2 as well as MMP-2 activity in two phases: In the initial phase, at 3 and 4 days, the mRNA of colligin and of collagen I and III was elevated predominantly in the LV, MMP-2 and TIMP-2 mRNA, as well as TIMP-2 protein and MMP-activity were increased in both ventricles. The second phase, after 14 days, was characterized by a less pronounced increase in colligin, collagen I and III and in MMP-2 activity which occurred exclusively in the LV. Finally, long-term treatment with NE induced a 37% increase in interstitial fibrosis which was shown to occur exclusively in the LV after 14 days. CONCLUSION: NE treatment induced fibrosis exclusively in the LV which was associated with hypertrophy predominantly of the LV. The elevated MMP-2 activity seems to be necessary for the ECM to adapt to the enlargement of myocytes and to reduce overproduction of collagen.

Changes in extracellular matrix and in transforming growth factor beta isoforms after coronary artery ligation in rats.

Extensive myocardial remodeling occurs after transmural myocardial infarction (MI). The infarcted myocardium is being replaced by scar tissue after gradual resorption of the necrotic tissue. The remodeling process involves both synthesis and degradation of collagens as major components of the extracellular matrix (ECM). In the present study we have analyzed the time-dependent changes of the processes related to this fibrosis in the infarct area and in the non-infarcted left ventricle (LV) six hours to 82 days after occlusion of the left anterior descending coronary artery (LAD) in rats. We also examined whether changes occurred in the expression pattern of the transforming growth factor (TGF) beta isoforms, since this cytokine is known as powerful inductor of fibrosis. Elevation in colligin expression preceded the pronounced increase in mRNA expression of both type I and type III collagen after MI from day three onwards. The maximal increase in colligin protein in the infarct area coincided with the most pronounced expression of collagen I and collagen III mRNA expression. Also, the expression and activity of matrix metalloproteinases (MMPs) and of tissue inhibitor of matrix metalloproteinase (TIMP)-2 mRNA were increased predominantly in the infarct area. TGF beta(1)and TGF-beta(2)expression increased within the first days after MI, whereas TGF-beta(3)expression was elevated predominantly in the infarct area. This pronounced increase in TGF-beta(3)persisted up to 82 days and correlated positively with the parameters of ECM metabolism. Thus, the scar formation is an ongoing dynamic process in which TGF-beta(3)seems to play an active role in the complex ventricular remodeling.

Comitogenic effect of catecholamines on rat cardiac fibroblasts in culture.

OBJECTIVE: We studied the ability of norepinephrine and of other catecholamines to affect the proliferation of cardiac fibroblasts isolated from adult rat hearts. Furthermore, we investigated the possible adrenergic receptor involved in this process. METHODS: Norepinephrine (NE), phenylephrine (PE), isoproterenol (ISO), forskolin (FO), epidermal growth factor (EGF), platelet-derived growth factor AA (PDGF-AA) and specific inhibitors of the alpha(1)-, alpha(2)-, beta(1)- and beta(2)-adrenoceptors and of the protein kinase A (PKA) were applied to cardiac fibroblasts in culture. Cell number was measured by use of a Coulter Counter. Activation of the cAMP response element binding protein (CREB) was measured by Western blotting and subsequent use of a phospho-specific antibody. Activation of the p42- and the p44-mitogen activated protein kinase (p42/p44(MAPK)) was assessed by detection of phosphorylation shifts and by incorporation of 32P-labelled phosphate into myelin basic protein. RESULTS: Fibroblasts isolated from hearts of adult rats were grown in 10% serum-containing media which induced an increase in cell number by 94%. After 48 h, treatment with 10 microM NE caused an even greater increase in cell number by 222%, i.e. another 128% (comitogenic effect). In contrast, NE alone had no effect on the growth of serum-deprived cells. EGF and PDGF-AA did not replace serum as the basic mitogen. After addition of NE to proliferating cells under serum conditions, there was a rapid, time-dependent significant activation of the p42/p44(MAPK) and of CREB for up to 60 and 120 min, respectively. In both cases, the maximum of activation was reached after 5 min. Application of FO (0.1-20 microM) caused a strong activation of CREB, while no increase in the phosphorylation of the p42/p44(MAPK) was detected. Treatment with 20 microM FO led to an identical increase in cell number as application of NE. Specific blockade of PKA with RpcAMPS prevented the activation of CREB and also the comitogenic effect of FO as well as of NE. The alpha- and beta-adrenergic receptor blocker carvedilol (10 microM) normalized all NE-induced effects. Prazosin and yohimbine, inhibitors of alpha(1)- and alpha(2)-adrenoceptor activation, respectively, did not influence the NE-evoked increase in cell number. In contrast, the non-selective beta-adrenoceptor blocker propranolol (1 microM) completely suppressed the comitogenic effect of NE. A similar effect was obtained with the specific beta(2)-adrenoceptor blocker ICI 118,551 (5 microM), while the beta(1)-adrenoceptor blocker metoprolol did not influence the increase in cell number. CONCLUSIONS: NE elicits a comitogenic effect on cultured rat cardiac fibroblasts which is prevented by beta(2)-adrenergic blockade. The activation of CREB contributes to the increase in proliferation. The p42/p44(MAPK) which was also found to be activated by NE might as well be involved in the regulation of the comitogenic effect.

Differential remodeling of the left and right heart after norepinephrine treatment in rats: studies on cytokines and collagen.

Continuous intravenous infusion of norepinephrine norepinephrine (NE, 0.1 mg/kg/h) induced hypertrophy of the left ventricle (LV), but not of the right ventricle (RV) in rats, although RV systolic pressure (RVSP) was much more elevated than LVSP. After NE infusion, there was a time-dependent (20 min to 72 h) expression in the mRNA of interleukin (IL)-6 and IL-1 beta. The expression of IL-6 increased markedly and reached the maximum after 4 h with an 80-fold elevation. In the RV, the expression increased only 20-fold. The mRNA of IL-1 beta increased significantly after NE stimulation only in the LV and reached the maximum after 12 h with a 12-fold elevation. After 12 h of NE infusion, colligin mRNA was elevated for the first time with further progression until 72 h. The six-fold abundance of colligin mRNA seen after 72 h was significantly higher in the LV than in the RV. A similar increase was observed on the protein level (Western blotting). The expression of collagen I and III increased significantly after 24 h only in the LV. After 72 h, the mRNA expression of collagen I was increased 16-fold and that of collagen III 10-fold. This expression was significantly higher than that in the RV. Also the elevation in atrial natriuretic peptide (ANP) mRNA started earlier and was more pronounced in the LV than in the RV. The alpha- and beta-adrenergic receptor blocker carvedilol normalized all functional parameters after 6 h and 72 h and prevented the development of LV hypertrophy that occurred after 72 h. The NE-induced increased expression of the mRNAs studied was either prevented (IL-6, IL-1 beta ) or attenuated (colligin, collagen I and III, ANP) by combined alpha- and beta-receptor blockade. The elevation of afterload which was associated with the NE effect was normalized by the calcium-channel blocker nisoldipin, but NE-induced LV hypertrophy and the increase in ANP and collagen mRNA were not affected.

Late signals from the PDGF receptors leading to the activation of the p70S6-kinase are necessary for the transition from G1 to S phase in AKR-2B cells.

Platelet-derived growth factor AB (PDGF-AB) has to be permanently present in the culture medium to achieve full proliferation (>90%) of AKR-2B fibroblasts. Upon removal after 1 h incubation time, only a small number of cells (<20%) entered the cell cycle. Concomitantly there was no increase in RNA- and protein-synthesis. The PDGF-receptor autophosphorylation reached a maximum after 30 min incubation with PDGF-AB. Tyrosine phosphorylation was no longer detectable after 2-4 h. The clustering of receptors into coated pits, analyzed by indirect immunofluorescence using a specific antibody against PDGF-beta-receptor, showed in contrast to autophosphorylation a biphasic kinetic. A first maximum was reached after 30 min, followed by a complete disappearance of coated pits, which regenerated in a second phase after 3 h and were long lasting. If PDGF-AB was removed after 1 h, the second phase was obliterated. The involvement of two different signalling pathways in these two phases was investigated in detail: (1) The ras-raf-MAP-kinase pathway and (2) the PI-3-kinase/p70(S6)-kinase pathway. PDGF-AB addition caused a fast (10 min) activation of MAP-kinase, which returned to background level after 1 h without any further activation later on. In contrast PDGF-AB led to a rapid (15-30 min) activation of the p70(S6)-kinase that persisted for 8-12 h just prior to the entry of the cells into S-phase. If PDGF-AB was removed after 1 h, the activation of this kinase ceased 3 h later. PDGF-AA, which is unable to promote division of AKR-2B cells, induced only a shortlasting p70(S6)-kinase activation. These observations add further evidence for the involvement of the p70(S6)-kinase pathway in the proliferation control of AKR-2B fibroblasts in the late G1 phase (4-8 h after growth factor addition). On the other hand, if the p70(S6)-kinase activation was prevented by the addition of 10 nM rapamycin, the cell division was not inhibited but only delayed by 4 h. Similar kinetics were observed when the PI-3-kinase was inhibited by 400 nM wortmannin. It is suggested that a regulatory element exists upstream of the p70(S6)-kinase and the PI-3-kinase. This regulatory element should be responsible for the transmission of late signals required for the progression through the cell cycle. This element is not involved in the immediate responses after PDGF-AB addition but must be stimulated within a second later phase of PDGF activation.

A 10-year experience in the use of air medical transport for medical scene calls.

OBJECTIVE: The objective of this retrospective descriptive study was to evaluate the use of air medical services in response to medical scene calls for transport to tertiary care in the rural setting. METHODS: This study is a retrospective descriptive review of all medical scene calls during a 10-year study period. The cases were analyzed for demographics, transport time, medical indication, procedures, role of ground EMS services, effects on community hospitals, and patient outcomes. A case-by-case review by emergency medicine (EM) physicians was conducted to determine necessity of air medical transport. RESULTS: A total of 8106 medical flights were conducted during the study period. Of these, 103 were scene calls for which 85 charts were available for review. The breakdown of medical scene calls is cardiac (29%), poisoning (17%), co poisoning (11%), neurologic (11%), and other (32%). Ground EMS was involved in 80% of the cases; ground advanced life support (ALS) was present in 58%. In 86% of the flights reviewed, an EM resident was aboard the helicopter. Of the 85 patients whose charts were available, 41 required admission to the ICU, five required hyperbaric oxygen (HBO) treatment, and 14 died before admission. CONCLUSION: Evacuation of the rural patient with a medical emergency accounts for an extremely small percentage of an air medical service's use. ALS services, including emergency procedures at the scene and rapid transport to a tertiary care, were provided. Seventy-one percent of the flights reviewed required transport to a tertiary care facility, indicating that air medical transport was appropriate. Physician guidelines to ensure effective and cost-efficient use of these services should be developed. Responding for victims in cardiopulmonary arrest appears to provide little benefit with respect to outcome.

Okadaic acid induces cellular hypertrophy in AKR-2B fibroblasts: involvement of the p70S6 kinase in the onset of protein and rRNA synthesis.

At low concentrations (50 nM), okadaic acid (OA), an inhibitor of phosphatases 1 and 2A, inhibits platelet-derived growth factor-induced cell proliferation in late G1 (A. Simm et al., Exp. Cell Res., 210: 160-165, 1994). This inhibition is caused by the interference of OA in the induction and activation of the cell division protein kinases cdk1 and cdk2. OA alone has no effect on cell number, but induces a pronounced increase in cell size. The OA-induced hypertrophy can be divided into two phases. The first phase is characterized by a swelling of the cells. This increase in cellular volume is not accompanied by a change in the level of cellular macromolecules, i.e., protein and RNA. Inhibitor studies indicated a possible role of the Na+/H+ antiporter and Cl- channels in this process. In the second phase, an increase in the cellular protein and RNA content was observed along with a minor change in cell volume. To delineate a possible signaling pathway, the involvement of numerous protein kinases was analyzed. Low concentrations of OA lead to pronounced and sustained activation of the p70S6 kinase. There was little or no effect on various other kinases that can be activated by extracellular signals, e.g., mitogen-activated kinase, ribosomal S6 kinase, or other S6 peptide kinases. Likewise, at these concentrations, OA did not activate the genes for fos, myc, or ornithine decarboxylase. At very low concentrations (ED50, 0.5 nM), rapamycin, a specific inhibitor of the activation of p70S6 kinase, reversed the activation of the p70S6 kinase and the enhancement of RNA synthesis and partially the increase in cell volume and protein synthesis. The OA-induced hypertrophy of AKR-2B fibroblasts may serve as a model system for investigations aimed at the identification of signaling pathways leading to hypertrophy of differentiated nonproliferating cells.

[Treatment of anemia in pregnant women with an animal blood preparation, "Livex"]. / Leczenie niedokrwistosci kobiet ciezarnych preparatem z krwi zwierzecej pod nazwa "Livex".

The animal blood preparation Livex obtained from the Agricultural Academy in Wroclaw was used for treatment of anaemia of pregnancy. During four weeks of treatment (3 x 7.0 g of Livex added to diet) a statistically significant increase was found of anaemia parameters such as haemoglobin level, value of haematocrit, red blood cell count, and mean corpuscular volume. Other parameters such as mean corpuscular haemoglobin mass, and mean corpuscular haemoglobin concentration were also increased but statistically insignificantly. Livex is an effective and safe preparation causing no side effects in view of the presence of certain trace elements in it, especially iron, copper, zinc, cobalt, and selenium. The use of this preparation in the treatment of anaemia of pregnancy is a natural and effective method of erythropoietic process stimulation.

Air medical tracheal intubation: establishing a threshold for this QA indicator.

Endotracheal intubation is a critical skill necessary in a number of situations encountered by air medical personnel. The purpose of this study was to establish a threshold for the quality assurance indicator of successful tracheal intubation in a physician-staffed air medical system. The records of all patients transported by a physician-staffed air medical system over a 36-month period were reviewed. One hundred and forty-three patients had endotracheal intubation attempted. Blind nasotracheal intubation attempts were successful in 71% of those in whom it was attempted, while the overall intubation success rate was 92%. Based on this study and the existing literature, a threshold of 90% is recommended for the quality assurance indicator of successful tracheal intubation in physician-staffed air medical systems.

The decision to add a second hospital-based EMS helicopter.

An analysis of the first seven years of performance of our hospital-based emergency medical services (EMS) helicopter was conducted to evaluate the possible need for a second aircraft. A survey of seven hospitals currently operating two or more helicopters resulted in a consensus that one helicopter can effectively perform only 70 to 90 flights per month. The number of requests for our helicopter service has increased 148% from 610 to 1,512 in seven years while the number of completed missions has increased only 92% from 486 (40.5/month) to 935 (78/month). Requests denied due to inclement weather (265 in 1988) cannot be captured with a second visual-flight-rated (VFR) EMS helicopter; however, those missed due to maintenance requirements of the helicopter and overlapping requests (232 in 1988) can be captured. The need for a second aircraft exists when the number of requests for the service grows while the number of captured flights plateaus. Our data and industry survey suggests this will occur at 75 captured flights per month. Affordability and continued overall growth of trauma and other critical care referrals to the base hospital(s) is mandatory. This study provides a model for hospital-based EMS helicopter operators to apply to the decision whether to add a second aircraft.

Cricoarytenoid arthritis: a cause of laryngeal obstruction.

Cricoarytenoid arthritis, principally as a result of long-standing rheumatoid arthritis, is a disorder that may present to the emergency physician with a number of symptoms and signs referable to the larynx. The presentation of this disorder as acute laryngeal obstruction and collapse is uncommon. It is important to recognize early cricoarytenoid joint involvement and not mistakenly diagnose mild cases as asthma or psychoneurosis. The use of steroids in mild cases has been beneficial at times, but progressive airway obstruction and fatalities have occurred during their administration.

Physician-staffed helicopter scene response from a rural trauma center.

One hundred ten consecutive trauma patients transported directly from the scene of injury by a hospital-based helicopter emergency medical service were reviewed. These patients were injured in the rural areas of central and northeastern Pennsylvania and were transported to a level I trauma center. The medical flight team consisted of an emergency physician and a registered nurse. The average round-trip flight distance was 44 miles. The overall mortality was 21.8%. Eighty-eight percent of the injuries were motor vehicle related. Only two patients received advanced life support prior to arrival of the helicopter. Forty-five percent of patients required major intervention by the medical flight team at the scene. The average scene time was 33.6 minutes for transported patients. Sixteen patients required extrication after the arrival of the flight crew and had significantly prolonged scene times averaging 61.8 minutes (P less than .001). Patient entrapment was the most important contributing factor in on-scene ground time.

Rural interhospital helicopter transport of motor vehicle trauma victims: causes for delays and recommendations.

One hundred twenty-six consecutive ACS Category I motor vehicle trauma patients transported by helicopter from 25 hospitals to a regional trauma center in rural Pennsylvania during a 14-month period were reviewed retrospectively. The overall mortality was 13%. Average round-trip distance was 79 miles. Interventions by the medical flight team (emergency physician/nurse) included endotracheal intubation, tube thoracostomy, and/or central venous access in 42 patients (33%) prior to lift-off. Ground time at the referring facility, from landing to lift-off, when no interventions were required of the flight team, averaged 31.2 minutes (baseline). Ground time when major therapeutic interventions were required (principally airway management), however, averaged 57.4 minutes, an 84% increase over baseline (P less than .01). A major cause of the excessive ground times was the lack of standardized diagnostic workup and stabilization of patients prior to arrival of the flight team. Recommendations for standardized emergency department preparation of trauma victims requiring aeromedical evacuation are made.

Hospital-based rotorcraft aeromedical emergency care services and trauma mortality: a multicenter study.

A group of 1,273 blunt trauma patients who were treated and transported from the site of injury by seven different hospital-based rotorcraft aeromedical emergency care services were studied using a methodology based on injury severity designed to predict the mortality of such patients. The methodology predicted that 241 patients should have died; 191 patients did die. This 21% reduction in expected mortality was highly significant (P less than .001). Each of the seven rotorcraft services had a reduction in predicted mortality. The reduction was statistically significant (P less than .05) in five of the seven aeromedical services, or 86% of the total patient cohort. Hospital-based rotorcraft aeromedical emergency care services may reduce the expected mortality of blunt trauma patients treated at the site of injury.