Microfluorimetric quantification of catecholamine fluorescence in rat sympathetic ganglia

Summary The formaldehyde-induced fluorescence (FIF) technique was used to generate catecholamine fluorphores in the perikarya of the sympathetic neurons in the superior cervical ganglion of adult rats. During microfluorimetric quantification, the photodecomposition was eliminated by a rapid measuring procedure with a small excitation field and by using only visible light between the measurements.The catecholamine fluorescence, induced in protein microdroplets with increasing noradrenaline concentrations, was linear up to 2×10^–2 M which exceeds the noradrenaline content of even the most intensively fluorescent neurons. Thus, the differences in fluorescence intensities directly reflect the physiological state of each neuron with respect to their catecholamine content. The mean histograms reveal the changes which can only occur in certain neurons, and which can disappear if the mean only is assessed. The microfluorimetric method was sensitive enough to detect even minute changes induced by reserpine treatment in the catecholamine content of the sympathetic ganglion cells.     

Interstitial noradrenaline concentration of rat hearts as influenced by cellular catecholamine uptake mechanisms

Marked concentration differences of noradrenaline (NA) between the vascular and the interstitial compartment were detected by sampling interstitial transudate from isolated perfused rat hearts. The ratios of vascular/interstitial concentration amounted to 7.4 to 1.3 depending on the concentration of NA administered (3 × 10^–9 to 10^–6 M). These concentration differences were abolished by inhibitors of uptake₁ desipramine (DMI) I and uptake, (O-methyl-isoprenaline (OMI)). Neuronal uptake, was characterized by a K_m of 0.22 mol/l and a V_max of 370 pmol × min^–1 × gWWT^–1, extraneuronal uptake₂ by a K_UPTAKE of = 0.313 min^–4.The apparent permeability surface area (P×S)-product calculated from uptake rate and transcapillary concentration difference was significantly decreased by administrating 100 mol/l (NA) in presence of DMI. A presumed endothelial uptake mechanism contributing to catecholamine translocation was investigated in endothelial cells in culture. These cells showed a specific noradrenaline uptake with a K_m of 4.35 mol/l and a V_max of about 75 pmol × min^–1 x gWWT^–1. Any inhibiton by inhibitors of both of the two noradrenaline uptakes was lacking. The uptake rate of this mechanism is insufficient to contribute to the diffusive conductivity of the capillary wall (P × S-product). We conclude from our investigations on interstitial concentrations of catecholamines and transcapillary concentration differences, that the capillary wall, owing to its metabolic and diffusional characteristics, influences the exchange of catecholamines to a substantial and physiologically relevant extent.     

Regulation of phagocytic process of macrophages by noradrenaline and its end metabolite 4-hydroxy-3-metoxyphenyl-glycol. Role of α- and β- adrenoreceptors

The regulatory capacity of noradrenaline and its end metabolite 4-hydroxy-3-metoxyphenylglycol (HMPG) on the complete phagocytic process of macrophages were investigated. Either noradrenaline or HMPG did not modify adherence. However, 10^–12 M of noradrenaline stimulated the chemotaxis of macrophages, mainly mediated by -adrenergic receptors. In contrast, 10^–12 M of HMPG induced an opposed effect on this stage of the phagocytic process. To stimulate phagocytosis, it is necessary to employ a higher concentration (10^–5 M) of noradrenaline and this effect was blocked with either 10^–6 M propranolol or 10^–6 M phentolamine, and maintained by HMPG. Noradrenaline and HMPG did not modify the microbicide capacity of macrophages (measured by O₂ ^– production after phagocytosis). In conclusion, noradrenaline modulates the phagocytic process of macrophages, and this modulation is completed by HMPG, maintaining the phagocytic functions at physiologically optimal levels. Modulation of chemotaxis is mainly mediated by a-receptors and phagocytosis needs both - and -receptor-stimulation.     

The effects of microtubule and microfilament disrupting agents on cytoskeletal arrays and wall deposition in developing cotton fibers

Summary The effects of various cytoskeletal disrupting agents (cholchicine, oryzalin, trifluralin, taxol, cytochalasins B and D) on microtubules, microfilaments and wall microfibril deposition were monitored in developing cotton fibers, using immunocytochemical and fluorescence techniques. Treatment with 10^–4 M colchicine, 10^–6 M trifluralin or 10^–6 M oryzalin resulted in a reduction in the number of microtubules, however, the drug-stable microtubules still appear to influence wall deposition. Treatment with 10^–5 M taxol increased the numbers of microtubules present within 15 minutes of application. New microtubules were aligned parallel to the existing ones, however, some evidence of random arrays was observed. Microtubules stabilized with taxol appeared to function in wall organization but do not undergo normal re-orientations during development. Microtubule disrupting agent had no detectable affect on the microfilament population. Exposure to either 4×10^–5 M cytochalasin B or 2×10^–6M cytochalasin D resulted in a disruption of microfilaments and a re-organization of microtubule arrays. Treatment with either cytochalasin caused a premature shift in the orientation of microtubules in young fibers, whereas in older fibers the microtubule arrays became randomly organized. These observations indicate that microtubule populations during interphase are heterogeneous, differing at least in their susceptibility to disruption by depolymerizing agents. Changes in microtubule orientation (induced by cytochalasin) indicate that microfilaments may be involved in regulating microtubule orientation during development.     

Activation of the cAMP-generating system by vasoactive intestinal polypeptide (VIP) in the human laryngeal malignant cell line HEp-2

In the presence of 3-isobutyi-l-methylxanthine, VIP produced a dose-related (3×10^–9–10^–7 M) increase (g-fold) in cAMP production in isolated HEp-2 cells incubated at 15°C in KRP buffer. Among the peptides structurally related to VIP, including secretin (10^–7 M), pancreatic glucagon (10^–6 M), PHI, somatostatin-14 (10^–6 M), hpGRF (10^–8–4×10^–M), GIP (2×10^–7 M), only PHI (3×10^–7 M and above) is able to activate the cAMP-generating system in HEp-2 cells, but at 102 times lower potency. Under the same conditions, histamine (10^–3 M) was also ineffective, while PGE 2 (10^–7–10^–4 M) increased (0-fold) basal cAMP levels in HEp-2 cells. The VIP effect is related to the interaction os the peptide on VIP recognition sites (12SI-VIP-binding capacity ), coupled to the membrane-bound adenylate cyclase . The results indicate that the transformed laryngeal cell line HEp-2 possessesa receptor-cAMP system preferentially activated by VIP (relative potencies: VIP > PHI other peptides of the secretin family), and suggest that this neuropeptide could modulate biological functions in normal laryngeal epithelia in man.     

Paraganglia in the urogenital tract of man

Summary According to the earlier concept, the paraganglia of man are believed to degenerate during the first postnatal years after their dominance during the fetal period. Clinical case reports on persisting paraganglia led us to extensive exploration of surgical material obtained from urological and gynecological surgery. The formaldehyde induced fluorescence (FIF) was used for tracing the catecholamine containing tissues. The fluorescence intensities were recorded with a Lietz MPV 2 microspectrophotometer.Solitary, small paraganglia were found in all patients studied. They were expecially frequent in the walls of the urinary bladder and in the connective tissue surrounding the urogenital organs. The intensity of the fluorescence was comparable to pharmacological standard of 10^–2 M noradrenaline and at the same level as the FIF of human fetal paraganglia. All cells of the paraganglionic clusters exhibited FIF and no signs of degeneration could be observed.It is suggested that the paraganglia of man do not degenerate postnatally but persist as a remarcable catecholamine reservoir, which might be of physiological importance.     

The role of cellulase in hormonal regulation of shoot morphogenesis in tobacco callus

A polyclonal antibody raised against cellulase (EC 3.2.1.4.) from callus ofNicotiana tabacum L. cv. Petit Havana SR1 reduced cellulase activity and induced shoot formation in tobacco callus in the presence of callus maintaining concentrations of auxin and cytokinin. Shoot induction as well as reduction of the cellulase activity was also obtained by withdrawing auxin from the callus medium. The effect of the two hormones on cellulase activity in the tobacco tissue was examined by varying the concentration of one of the hormones -naphthylacetic acid (NAA) or benzylaminopurine (BAP) at a time while the other was kept at a level sufficient for either callus growth or shoot induction. While NAA stimulated the enzyme activity increasingly with concentration in the range 5 × 10^–7 M to 5 × 10^–5 M at both levels of BAP, BAP only stimulated the cellulase activity at an optimum concentration of 5 × 10^–6 M when NAA was present at a level sufficient to induce callus growth. The results point to a pivotal role of the downward regulation of cellulase in the initiation of shoot induction. A series of events leading to oriented cell divisions as a result of the lowered cellulase level during the initial phase of the morphogenetic process is discussed.Abbreviations Ab Purified cellulase antibody - BAP benzylaminopurine - MS Murashige and Skoog medium - NAA -naphthylacetic acid - PS Purified preimmune serum We thank Mr. Poul Fabech for constructing the automatic viscosimetric equipment and Mr. Hans Hjorth for making the computer programme. This work was supported by The Danish Veterinary and Agricultural Research Council.     

Activating effect of substance P on nerve tissue in culture

The effect of substance P on explant development was investigated in organotypic cultures of rat sympathetic ganglia and spinal cord. The pattern of evolution, cellular composition, and dimensions of the growth zone were evaluated on the basis ofin vivo observations. It was found that this peptide exercises a significant growth-promoting effect at a concentrations of 10^–5–10^–12 M for sympathetic ganglia and 10^–5–10^–14 M for spinal cord culture. The growth zone of sympathetic ganglia measured 1.3–1.6 times the control level by the 14th day of culture at all effective concentrations. The area of outgrowth of spinal cord explants increased 2.0–5.2 fold by the sixth day of culture and peak response was recorded at concentrations of 10^–5 and 10^–12 M. This effect resembled response to opioid peptides [1, 3]. The likely physiological significance of regulatory peptides for the processes of nerve tissue development and regeneration is discussed in the light of these findings, together with the part played by the nociceptive/antinociceptive system in processes of histogenesis and repair.Institute of Experimental Cardiology, All-Union Cardiologic Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 610–615, September–October, 1986.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Energy transfer to the reaction centres in bacterial photosynthesis

From a combined study of (1) bacteriochlorophyll fluorescence lifetimes, (2) relative yields and (3) differential absorption changes corresponding to the reaction centres photooxidation, the absolute values of fluorescence lifetimes and quantum yields for two bacteriochlorophyll fractions have been calculated. The main bacteriochlorophyll fraction (80–90%) serving as a light-gathering antenna for reaction centresP ₈₉₀ is characterized by dark values of fluorescence lifetimes of the order of 10^–11 sec and fluorescence yields of 10^–3. The remaining part of the bulk pigment, not associated withP ₈₉₀ as far as excitation energy transfer is concerned, has an approximately constant fluorescence yield of about 5–8% and lifetime of about 10^–9 sec. Basing on these results, excitation jump times and intermolecular coupling energies were estimated to be 10^–13 sec and 10^–2 ev respectively. The conclusion is made that excitation energy transfer in the main part of bacteriochlorophyll occurs by the exciton mechanism at moderate intermolecular energies.     

Inhibitory effects of GABA on synaptic excitation in isolated rat hippocampal slices

In research on -aminobutyric acid (GABA) used at different concentrations on the amplitude of EPSP within populations (PEPSP), as recorded from dentrites in isolated hippocampal slices, GABA induced a dose-dependent reversible reduction in PEPSP amplitude with no noticeable signs of desensitization. Highest sensitivity to GABA was shown by PEPSP in hippocampal zone CA1 (threshold concentration: 3×10^–5–2×10^–4 M; (concentration at which the effect equal to 1/2 of maximum occurs) IC₅₀: 5×10^–4–1×10^–3 M). The effects of GABA on PEPSP were not blocked by bicuculline, picrotoxin, or penicillin. Action of GABA on dendritic antidromic population spike (DAPS — postynaptic effects) were slightly diminished by these blockers. Baclofen inhibited PEPSP more powerfully than GABA (threshold concentration: 1×10^–6 M: IC₅₀: 3×10^–6 M), although it only produced a minor reduction in DAPS amplitude even at high concentrations. It is concluded that the inhibitory effect of GABA on PEPSP in hippocampal zone CA1 may be put down mainly to its presynaptic action mediated by GABA_B receptors on axonal terminals of Schaffer collaterals.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 627–633, September–October, 1990.     

Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary The localization of l-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10–20 m in diameter and formed clusters or occured as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occured TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Noradrenaline and its end metabolite 3-methoxy-4-hydroxyphenylglycol inhibit lymphocyte chemotaxis: Role of alpha- and beta-adrenoreceptors

The capacity of noradrenaline (NA) and its end metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) to modulate the chemotaxis of lymphocytes from a primary immunocompetent organ (thymus) and a secondary one (spleen) was investigated over a range of concentrations from 10^–12 M to 10^–5 M. Lymphocyte chemotaxis was evaluated in a Boyden chamber. The results indicated that 10^–5 M of NA inhibits the chemotaxis of lymphocytes from both the immunocompetent organs studied, and that this effect is blocked by either propranolol (10^–6 M) or phentolamine (10^–5 M). Similarly, 10^–5 M of MHPG induced a decrease in the chemotaxis capacity of the lymphocytes. In conclusion, high physiological concentrations of NA and its end metabolite modulate the mobility of lymphocytes, and the participation of both alpha and beta adrenoreceptors is necessary, showing a new aspect of neuroimmune interactions.     

Growth and uptake kinetics of a facultatively oligotrophic bacterium at low nutrient concentrations

In oligotrophic waters, not only community structure but also physiological properties of heterotrophic bacteria are influenced by the concentration of organic matter.The relationship between growth rate of two facultatively oligotrophic strains ofAeromonas sp. No. 6 andFlavobacterium sp. M1 was studied in comparison with that of two eutrophic strains ofEscherichia coli 7020 andFlavobacterium sp. M2. These strains had two or three different substrate constants (Ks values) depending on substrate concentrations: Ks values for the two former were remarkably lower than those for the two latter. For instance, Ks value forAeromonas sp. No. 6 was about 8.9M when substrate concentration was greater than 53M and about 1.1M when substrate concentration was less man 53M. InE. coli the Ks value was about 260M at greater than 5600M and about 47M at less than 5600M substrate concentration.Uptake kinetics ofAeromonas sp. grown in a medium containing 2.7 mM glutamate (H-cell) and 0.11M glutamate (L-cell) have been determined for the intact cells. H-cell had two distinct values of Km for glutamate assimilation and respiration, and L-cell had three distinct values of Km for glutamate assimilation and respiration: In H-cell Km of assimilation was 2.8×10^–7 M and 1.5×10^–4 M, and Km of respiration was 2.3×10^–7 M and 1.7×10^–4 M; in L-cell Km of assimilation was 7.4×10^–8 M, 8.3×10^–6 M, and 1.3×10^–4 M, and Km of respiration was 2.5×10^–7, 8.9×10^–6M, and 1.7×10^–4 M. More than 60% of glutamate taken up by the H- and L-cells was respired when the substrate concentration was less than 10^–6 M, although at greater than 10^–6 M, 50% and 30% of glutamate was respired by H-cells and L-cells, respectively. These results suggest that the facultatively oligotrophic bacteria grow with high efficiency in environments with extremely low nutrient concentration, such as oligotrophic waters of lakes and ocean, as compared with in their growth in conditions of high nutrient concentraton, such as nutrient broth.     

Certain withanolides from Iochroma gesnerioides antagonize ecdysteroid action in a Drosophila melanogaster cell line

Sixteen withanolides isolated from Iochroma gesnerioides (Kunth) Miers (Solanaceae) have been assessed for their activities as ecdysteroid agonists and antagonists. None of the compounds showed any agonistic activity, but several showed significant antagonistic activity. With a 20-hydroxyecdysone concentration of 5 × 10^–8 M, the ED₅₀ values for 2,3-dihydro-3-methoxywithaferin A, 2,3-dihydro-3-methoxywithacnistine, 2,3-dihydro-3-methoxyiochromolide and 2,3-dihydro-3-hydroxywithacnistine are 3.5 × 10^-5 M, 1 × 10^–5 M, 5 × 10^–6 M and 2.5 × 10^–6 M, respectively.     

Ca2+-dependent phosphorylation by endogenous kinases of Mr 95 K and 50 K-55 K proteins in PC12 pheochromocytoma cells

Endogenous protein phosphorylation of PC12 cells was investigated with the homogenate as well as intact cells. In the case of the homogenate, the major proteins that were phosphorylated in the presence of Ca²⁺ were found to be of Mr 95 K and Mr 50 K-55 K. Ca²⁺/calmodulin-dependent protein kinase appeared to be responsible for phosphorylation of Mr 50 K-55 K proteins and partly of Mr 95 K protein. The apparentK _m's for Ca²⁺ of Mr 95 K and 50 K-55 K protein phosphorylation were 2.2×10^–7 M and around 1.5×10^–6 M, respectively. Since several cell lines of neuroblastoma exhibited Mr 95 K protein phosphorylation of similar type, the protein phosphorylation may be a common process shared by neuronal cells. Depolarization of intact PC12 cells by high K⁺ concentrations induced Mr 95 K protein phosphorylation. The results suggest that a physiological increase by excitation in the intracellular Ca²⁺ concentration triggers phosphorylation of Mr 95 K protein in neuronal cells and this phosphorylation may play a role in the regulation of transmitter release.     

In vitro propagation of the bamboo (Bambusa tulda Roxb.) through shoot proliferation

An efficient protocol has been developed for the in vitro propagation of Bambusa tulda through shoot proliferation. Shoots from 3-week-old aseptically grown seedlings were used to initiate cultures. Multiple shoots were obtained on liquid Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (8×10^–6M) and kinetin (4×10^–6M). Continuous shoot proliferation at a rate of 4–5 fold every three weeks was achieved through forced axillary branching. More than 90% of the shoots could be rooted on a modified MS medium containing indoleacetic acid (1×10^–5M) and coumarin (6.8×10^–5M). Following simple hardening procedures, the in vitro raised plants were transferred to the soil with more than 80% success.Abbreviations BAP 6-benzylaminopurine - 2-ip 6-,-dimethylallylaminopurine - Kn kinetin - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - NAA 1-naphthaleneacetic acid     

Hydrocortisone-induced increase in the number of small intensely fluorescent cells and their histochemically demonstrable catecholamine content in cultures of sympathetic ganglia of the newborn rat

Synopsis It is known that hydrocortisone causes a great increase in the number of small intensely fluorescent (SIF) cells in the sympathetic ganglia when injected into newborn rats. The effect of hydrocortisone on nervous tissuein vitro has not been studied previously.Pieces of newborn rat sympathetic ganglia were cultivated in Rose chambers. Hydrocortisone was dissolved in the medium in concentrations of 1–9 mg/l. Both control and hydrocortisone-containing cultures were examined daily by phase-contrast microscopy, and the catecholamines were demonstrated histochemically by formaldehyde-induced fluorescence after 7 days in culture.All cultures showed outgrowths of axons and supporting cells elements, although these were less extensive in the groups of cultures with hydrocortisone. After a week, SIF cells with a green fluorescence were observed in the control explants. In all cultures with hydrocortisone, a concentration-dependent increase was observed in the fluorescence intensity and the number of the SIF cells in the explant; numerous SIF cells were also seen in the outgrowth. Some SIF cells showed processes and the longest processes were seen in cultures with the highest concentration of hydrocortisone.It is concluded that hydrocortisone causes an increased synthesis of catecholamines in the SIF cellsin vitro, and an increase in their number by affecting either their division or their differentiation from a more immature form, or both. This effect was a direct one and not mediated by any system other than the ganglion itself. Induction of enzyme synthesis by hydrocortisone is proposed as an explanation of the increase in catecholamine concentration.University of Melbourne Senior Research Fellow, September 1971-August 1972Sunshine Foundation and Rowden White Trust Overseas Research Fellow in the University on Melbourne, September 1971-August 1972     

Simultaneous determination of cholecystokinin,dopamine, glutamate and aspartate in cortex and striatum of the rat using in vivo microdialysis

Summary Extracellular levels of cholecystokinin (CCK), dopamine (DA), glutamate (Glu) and aspartate (Asp) were simultaneously monitored in the frontoparietal cortex and the striatum of halothane-anaesthetized rats using in vivo microdialysis. Under basal conditions, cortical and striatal CCK levels were 3.11 ± 0.39 pM and 2.76 ± 0.15 pM, respectively. Local KCl (10^–1 M) and bicuculline (10^–4 M) co-application in cortex or striatum increased the CCK levels 18-fold and 26-fold, respectively. The DA level in striatum was 3.78 ± 0.28 nM and the local perfusion with KCl + bicuculline led to a 45-fold increase. The cortical and striatal outputs of Glu were of the order of 2 · 10^–6 M and Asp levels were around 6 · 10^–7 M. Local stimulation with KCl (10^–1 M) and bicuculline (10^–4 M) caused a small increase (2 fold) in cortical and striatal levels of Glu and Asp. The addition of KCl (10^–1 M) and bicuculline (10^–4 M) to the cortical perfusion medium did not modify CCK, DA or Glu concentrations in striatum. These results demonstrate that CCK, DA, Glu and Asp may be simultaneously monitored in vivo and support the idea that their extracellular levels recovered in the microdialysis perfusates could be derived from neuronal pools.     

Survival,morphology, and catecholamine storage of chromaffin cells in serum-free culture: Evidence for a survival and differentiation promoting activity in medium conditioned by purified chromaffin cells

Adult bovine and young rat chromaffin cells cultured in serum-free medium were examined for their survival and differentiation following exposure to various additives, trophic agents and conditioned media. Adrenal chromaffin cells dissociated from 8 day old rats were maintained by dexamethasone, NGF and CNTF or without any additives in an N1-supplemented medium in similar numbers as in serum-containing medium for up to 6 days. Neuritic growth elicited by NGF or CNTF was enhanced in the absence of serum. Medium conditioned by purified bovine chromaffin cells improved cell survival and caused neurite outgrowth in a dose-dependent manner. The activiti(es) was sensitive to heat and trypsin and not blocked by the addition of anti-NGF antibodies. Bovine chromaffin cell survival was reduced by 30% when cells were maintained for one week in the absence as compared to the presence of serum. Addition of insulin, the N1 supplement, dexamethasone or dbcAMP single or in combinations improved the survival to different extents. A combination of insulin (5 g/ml) and dexamethasone (5×10^–6M) proved to be optimal in this respect. However, these supplements failed to restore the cellular catecholamine, noradrenaline and adrenaline contents to levels seen in the presence of serum. This was also true for a chromaffin cell-conditioned medium, which improved survival without elevating the catecholamine contents. Conditioned medium, however, partly restored a more physiological adrenaline-noradrenaline-ratio.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).