Calcium ion uptake, somatotropin release, and fine structure of somatotrophs in cultures of the rat anterior pituitary upon the action of an oligopeptide (Boc-Gln-Leu-Lysinal)

Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, Boc-Gln-Leu-Lys-H. The oligopeptide had a profound releasing effect on growth hormone, whereas the prolactin release remained unchanged at 10(-3) mol/l drug concentration after an incubation for 2 h. In the presence of the oligopeptide a time- and dose-dependent calcium influx into cultured cells has been shown which was proved to be almost completely antagonized with magnesium ions but not with Nifedipine. In addition, radioactive calcium ions could be detected in a number of cells by light microscopic autoradiography when cultures were treated with Boc-Gln-Leu-Lys-H for short periods. The selective Gel action of the oligopeptide on growth hormone producing cells has been demonstrated also in fine structural investigations: multigranular and single exocytotic profiles have been observed. Accordingly, we have postulated that Boc-Gln-Leu-Lysinal mimics the effects of the known ionophores. Its mode of action needs, however, further studies especially on isolated somatotrophs.     

Voltage gated calcium channels in molluscs: classification,Ca2+ dependent inactivation,modulation and functional roles

Molluscan neurons and muscle cells express transient (T-type like) and sustained LVA calcium channels, as well as transient and sustained HVA channels. In addition weakly voltage sensitive calcium channels are observed. In a number of cases toxin or dihydropyridine sensitivity justifies classification of the HVA currents in L, N or P-type categories. In many cases, however, pharmacological characterization is still preliminary. Characterization of novel toxins from molluscivorousConus snails may facilitate classification of molluscan calcium channels. Molluscan preparations have been very useful to study calcium dependent inactivation of calcium channels. Proposed mechanisms explain calcium dependent inactivation through direct interaction of Ca²⁺ with the channel, through dephosphorylation by calcium dependent phosphatases or through calcium dependent disruption of connections with the cytoskeleton. Transmitter modulation operating through various second messenger mediated pathways is well documented. In general, phosphorylation through PKA, cGMP dependent PK or PKC facilitates the calcium channels, while putative direct G-protein action inhibits the channels. Ca²⁺ and cGMP may inhibit the channels through activation of phosphodiesterases or phosphatases. Detailed evidence has been provided on the role of sustained LVA channels in pacemaking and the generation of firing patterns, and on the role of HVA channels in the dynamic changes in action potentials during spiking, the regulation of the release of transmitters and hormones, and the regulation of growth cone behavior and neurite outgrowth. The accessibility of molluscan preparations (e.g. the squid giant synapse for excitation release studies,Helisoma B5 neuron for neurite and synapse formation) and the large body of knowledge on electrophysiological properties and functional connections of identified molluscan neurons (e.g. sensory neurons, R15, egg laying hormone producing cells, etc.) creates valuable opportunities to increase the insight into the functional roles of calcium channels.     

The calcium-mediated promotion of mitotic activity in rat thymocyte populations by growth hormone, neurohormones, parathyroid hormone and prolactin

Growth hormone, oxytocin, parathyroid hormone, prolactin and lysine vasopressin strongly stimulate mitotic activity in rat thymocyte populations maintained in vitro. These hormones have no mitotic effect on cells maintained in calcium-free medium. It is concluded that they stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium. The stimulatory action of calcium itself is opposed by low concentrations of the mucopolysaccharide chondroitin sulphate. However, the inhibitory action of chondroitin sulphate can be overcome by growth hormone. A possible common mechanism of action of these hormones on mitotically competent cells is discussed.     

Calcium-dependent anion channel in the water mold,Blastocladiella emersonii

Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr²⁺ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr²⁺. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

Model for the Action of Calcium in Muscle

CALCIUM ions play an important part in contraction^1–3, but little is known of the way that changes in internal calcium actually control the events that lead to the development of tension. Most experiments involving calcium have been performed under steady state conditions at different stabilized calcium concentrations^4–9 and can only give limited information as to the action of calcium. Recently, tension transient experiments have been described at either constant calcium concentration^6,7 or when the calcium concentration was varying in a known manner^10,11. From these and related experiments, a model for the kinetics of calcium has been deduced which predicts not only the time course of tension development from just a knowledge of the free calcium concentration, but which is also able to correlate the ATPase, tension and calcium binding responses in the steady state. The model considers that two calciums act as separate effectors with a de-repressant action in the same functional unit.     

Calmodulin-binding drugs affect responses to cytokinin, auxin, and gibberellic Acid

Trifluoperazine, a phenothiazine tranquilizer, and tetracaine, a local anesthetic, have been found to inhibit a variety of plant hormone responses at concentrations compatible with their known inhibition of Ca²⁺-calmod-ulin-dependent enzyme activities. Among these responses are cytokinin-dependent betacyanin synthesis and increase in fresh weight in Amaranthus tricolor cotyledons, auxin-dependent increase in length of wheat coleoptile segments and gibberellic acid-dependent induction of α-amylase synthesis in barley aleurone layers. The reversibility of some of these inhibitory effects has been demonstrated, indicating that, up to a point, a generalized membrane destruction can be ruled out. The evidence, taken in conjunction with numerous examples from the literature showing calcium involvement in the action of all of the plant hormones, support a unifying theory of hormone action.     

Calcium-dioxolene complexes: Rate constants of pyrocatechol oxidation in the presence of Ca<Superscript>2+</Superscript>

The effect of calcium ions on the rate of pyrocatechol autoxidation at pH 9.0 has been studied by mathematical modeling. The effect of Ca²⁺ on the oxygen absorption rate has been studied, and a kinetic model has been suggested, which takes different stages of interaction of pyrocatechol and its radical form with oxygen into account. It has been shown that the prooxidant action of Ca²⁺ is related to an abrupt increase (approximately by three orders of magnitude) in the rate constant of comproportionation (reaction of chain branching and formation of o-semiquinonates) and a marked decrease (by two orders of magnitude, from 1.4 · 10⁷ to 0.6 · 10⁵ M⁻¹ s⁻¹) in the rate constant of disproportionation of o-semiquinones. The system can be used as a model for studying the prooxidant action of calcium ions.     

The significance of Ca2+ in the morphogenesis of Micrasterias studied with EGTA,verapamil, LaCl3 and calcium ionophore A 23187

The significance of Ca²⁺ in the morphogenesis of Micrasterias torreyi Bail. cells (grown in calcium-containing medium) was studied using the calcium chelating agent EGTA, the Ca²⁺ influx inhibitors verapamil and LaCl₃, and the calcium ionophore A 23187. In the experiments where the first three were used, it was possible to find a rather concise concentration area within which the growth and development of dividing cells were prevented, but where the cells remained alive and were capable of continuing their differentiation when moved into fresh medium. Cytoplasmic streaming and especially the cortical streaming system inside the cells was also disturbed or prevented during the action of these drugs. Higher concentrations caused the bursting of the treated cells, and even very low concentrations were able to inhibit the division of interphase cells. Ionophore A 23187 slowed down or prevented development, but, unlike the other drugs, accelerated the streamings and stimulated the division rhythm of interphase cells. The results support the hypothesis that Ca²⁺ influx through the plasma membrane affects and guides, during cell growth and development, the fusion of dictyosome vesicles, containing cell wall material, with the plasma membrane. They also indicate the importance of cortical cytoplasmic streamings in growth and differentiation, as well as the idea that the streaming is supported by actin microfilaments.     

Prolactin stimulates the L-type calcium channel-mediated transepithelial calcium transport in the duodenum of male rats

Elevated plasma levels of prolactin (PRL) have been reported in several physiological and pathological conditions, such as lactation, prolactinoma, and dopaminergic antipsychotic drug uses. Although PRL is a calcium-regulating hormone that stimulates intestinal calcium absorption in lactating rats, whether PRL is capable of stimulating calcium absorption in male rats has been elusive. Herein, the transepithelial calcium transport and electrical characteristics were determined in ex vivo duodenal tissues of male rats by Ussing chamber technique. We found that PRL receptors were abundantly present in the basolateral membrane of the duodenal epithelial cells. PRL (200–800 ng/mL) markedly increased the active duodenal calcium transport in a dose-dependent fashion without effect on the transepithelial resistance. The PRL-enhanced active duodenal calcium transport was completely abolished by L-type calcium channel blocker (nifedipine) as well as inhibitors of the major basolateral calcium transporters, namely plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger. Several intracellular mediators, such as JAK2, MEK, PI3K and Src kinase, were involved in the PRL-enhanced transcellular calcium transport. Moreover, PRL also stimulated the paracellular calcium transport in the duodenum of male rats in a PI3K-dependent manner. In conclusion, PRL appeared to be a calcium-regulating hormone in male rats by enhancing the L-type calcium channel-mediated transcellular and the paracellular passive duodenal calcium transport. This phenomenon could help restrict or alleviate negative calcium balance and osteoporosis that often accompany hyperprolactinemia in male patients.     

Transport of hepcidin, an iron-regulatory peptide hormone, into retinal pigment epithelial cells via oligopeptide transporters and its relevance to iron homeostasis

Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25 amino acids. This hormone binds to ferroportin, an iron exporter expressed on the cell surface, and facilitates its degradation. Here we investigated if hepcidin is a substrate for SOPT1 and SOPT2 and if the hormone has any intracellular function in RPE. Hepcidin inhibited competitively the uptake of deltorphin II (a synthetic oligopeptide substrate for SOPT1) and DADLE (a synthetic oligopeptide substrate for SOPT2) with IC₅₀ values in the range of 0.4–1.7 μM. FITC-hepcidin was taken up into RPE, and this uptake was inhibited by deltorphin II and DADLE. The entry of FITC-hepcidin into cells was confirmed by flow cytometry. Incubation of RPE with hepcidin decreased the levels of ferroportin mRNA. This effect was not a consequence of hepcidin-induced ferroportin degradation because excessive iron accumulation in RPE, which is expected to occur in these cells as a result of ferroportin degradation, did not decrease but instead increased the levels of ferroportin mRNA. This study reveals for the first time a novel intracellular function for hepcidin other than its established cell surface action on ferroportin.     

Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca²⁺) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca²⁺ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca²⁺-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca²⁺ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca²⁺ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca²⁺ pool in untreated cells when extracellular Ca²⁺ is low. However, low extracellular Ca²⁺ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca²⁺ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca²⁺ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Interaction of calcium ions with peptide hormones of the gastrin family

The interactions of Des-Trp¹-Nle¹²-minigastrin I (Nle¹¹-HG-13) and Nle¹⁵-little gastrin I (Nle¹⁵-HG-17) with calcium ions have been investigated in water and in trifluoroethanol solution using uv and CD absorption techniques. Both hormones strongly interact with Ca²⁺ in the organic medium. In the case of Nle¹¹-HG-13, the near-uv chiroptical properties (dominated by the transitions of the Trp residue in the C-terminal tetrapeptide sequence) indicate that three metal ions per mole of hormone are involved in the binding process. From the different response of near-uv and far-uv CD properties to the addition of calcium and from the change of the CD spectra in the aromatic absorption region, it is concluded that the biologically important C-terminal sequence is directly involved in the interaction with calcium. Elongation of the peptide chain from Nle¹¹-HG-13 to Nle¹⁵-HG-17 (Nle¹⁵-little gastrin I) does not provide any additional binding site for calcium ions. The change of the CD properties in the near- and far-uv indicates that three metal ions per mole of hormone are involved in the binding process. The dichroic absorption in the aromatic region indicates that only one of the two Trp residues of the little gastrin analog is sensitive to the presence of calcium. On the assumption that the variation of the CD properties is proportional to the extent of calcium binding, the binding constants K₁, K₂, and K₃ have been estimated roughly. Two similar sets of binding constants have been found, with K₁ ≥ 10⁶M^?1 and K₃ of the order of 10⁵M^?1, indicating similar binding sites in the two hormones with high affinity for calcium ions.     

Growth-dependent modulation of capacitative calcium entry in normal rat kidney fibroblasts

Normal rat kidney (NRK) fibroblasts have electrophysiological properties and intracellular calcium dynamics that are dependent upon their growth stage. In the present study we show that this differential behavior coincides with a differential calcium entry that can be either capacitative or non-capacitative. Confluent cells made quiescent by serum deprivation, which have a stable membrane potential near ? 70 mV and do not show spontaneous intracellular calcium oscillations, primarily exhibit the capacitative mechanism for calcium entry, also called store-operated calcium entry (SOCE). When the quiescent cells are grown to density-arrest in the presence of EGF as the sole polypeptide growth factor, these cells characteristically fire spontaneously repetitive calcium action potentials, which propagate throughout the whole monolayer and are accompanied by intracellular calcium transients. These density-arrested cells appear to exhibit in addition to SOCE also receptor-operated calcium entry (ROCE) as a mechanism for calcium entry. Furthermore we show that, in contrast to earlier studies, the employed SOCs and ROCs are permeable for both calcium and strontium ions. We examined the expression of the canonical transient receptor potential channels (Trpcs) that may be involved in SOCE and ROCE. We show that NRK fibroblasts express the genes encoding Trpc1, Trpc5 and Trpc6, and that the levels of their expression are dependent upon the growth stage of the cells. In addition we examined the growth stage dependent expression of the genes encoding Orai1 and Stim1, two proteins that have recently been shown to be involved in SOCE. Our results suggest that the differential expression of Trpc5, Trpc6, Orai1 and Stim1 in quiescent and density-arrested NRK fibroblasts is responsible for the difference in regulation of calcium entry between these cells. Finally, we show that inhibition or potentiation of SOCE and ROCE by pharmacological agents has profound effects on calcium dynamics in NRK fibroblasts.     

Occurrence, biosynthesis and properties of kinetin (N6-furfuryladenine)

In this paper we review the data on the structure and properties of N⁶-furfuryladenine (kinetin, K) accumulated during the last forty years. In 1955, kinetin was isolated from DNA as an artifactual rearrangement product of the autoclaving process. Subsequently, its cytokinin activity has been established, demonstrating a wide variety of biological effects, including those on gene expression, inhibition of auxin action, stimulation of calcium flux, the cell cycle, and as an anti-stress and anti-ageing compound. Recently, our views on this very well known plant hormone have changed. There are new data, which show that it occurs in cellular DNA as the product of oxidative, secondary modification and a secondary reaction of DNA. Also new results on the biological function of kinetin have been reported. Various biological effects produced by this hormone in vitro and in vivo have made kinetin even more scientifically interesting and commercially attractive as an ingredient of many beauty cosmetics.     

Towards the understanding of tetrahydroquinolines action in Aedes aegypti: larvicide or adulticide?

Aedes aegypti is an important vector of arboviruses such as dengue, yellow fever, chikungunya and Zika. Among the various types of insecticides used to combat this vector, the insect growth regulators have been developed and recommended for control of their larvae. In this work compounds with proven regulatory action, tetrahydroquinolines will be studied. These regulators act on the hormones responsible for the insect development. Ecdysone, one of the main hormones involved in this process has a specific receptor (EcR), where tetrahydroquinolines derivatives can bind, disrupting the normal action of this hormone, because they have structure similar to hormone 20-hydroxyecdysone (20E). In addition, studies show that this class of compounds interacts strongly in the potassium channel activated by calcium (BK channel). Thus, the goal is to study the action of compounds (tetrahydroquinolines) as insecticides and evaluate their larvicidal action (action on the ecdysone receptor) or adulticide (action on the BK channel) through homology modelling techniques, molecular docking and molecular dynamics simulations and aiming to propose a compound that presents both actions (larvicide / adulticide).     

The mode of action of venom from the endoparasitic wasp Pimpla hypochondriaca (Hymenoptera: Ichneumonidae) involves Ca+2‐dependent cell death pathways

The endoparasitoid Pimpla hypochondriaca injects venom during oviposition to condition its lepidopteran hosts. Venom is a complex mixture of proteins and polypeptides, many of which have been identified as enzymes, including phenoloxidase, endopeptidase, aminopeptidase, hydrolase, and angiotensin‐converting enzyme. Constituents of the venom have been shown to possess cytolytic and paralytic activity, but the modes of action of factor(s) responsible for exerting such effects have not been deciphered. In this study, we examined the mode of action of isolated venom using cultured cells (BTI‐TN‐5B1‐4). A series of blockage and inhibition assays were performed using a potent inhibitor (phenylthiourea, PTU) of venom phenoloxidase, and anti‐calreticulin antibodies. Monolayers exposed to venom alone were highly susceptible with more than 84.6±2.3% dead within 15 min. Susceptible cells displayed a retraction of cytoplasmic extensions, rounding, and swelling prior to lysis in more than half (55.7±1.7%) of the dying cells. Within 15 min of exposure to venom, cells displayed qualitative increases in [Ca⁺²]_i as evidenced by staining with the calcium‐sensitive probe fluo‐4 AM, and mitochondrial membrane potential (ΔΨ_m) was undetectable by 5 min post‐treatment with venom. These venom‐mediated changes occurred regardless of whether an external source of calcium was present, or whether venom was pre‐treated with PTU. In contrast, venom toxicity was attenuated by treatment with anti‐calreticulin antibodies. Not only did fewer cells die when exposed to antibody‐treated venom but also cell swelling diminished and no increases in intracellular calcium were detected. A possible mode of action for the venom is discussed. © 2009 Wiley Periodicals, Inc.     

Mechanism of action of benzo(a)pyrene and nicotine on hormone production by rat pituitary tumor cells

The effects of the cyclic aromatic hydrocarbon, benzo(a)pyrene (BaP) and that of the tobacco alkaloid, nicotine, on prolactin (PRL) and growth hormone (GH) synthesis by rat pituitary tumor cells in culture (GH cells) have been studied. Treatment of GH cells with nicotine (0.1–300 μg/ml) neither affected the growth, nor significantly altered the general pattern of hormone production in these cells. BaP at concentrations greater than 5 μg/ml irreversively inhibited the growth of these cells. The sublethal concentrations of BaP, which did not affect either 1) cell growth, or 2) amino acid transport or 3) total protein synthesis or degradation, did however inhibit specifically, hormone synthesis by these cells. More interestingly concentrations of nicotine which did not affect either cell growth or hormone synthesis, modulated both of these cellular processes in the presence of BaP. A concentration dependent stimulation of microsomal BaP monooxygenase activity was observed in nicotine or BaP treated cells. The effects of these drugs on stimulation of BaP monooxygenase activity seems to be additive. Nicotine also enhanced the association of radioactivity (presumably [³H] BaP metabolites) with DNA in [³H] BaP treated cells. It is concluded that nicotine by itself did not demonstrate any cytotoxic effect nor influence hormone synthesis in GH cells. However, this constituent of tobacco smoke stimulated BaP monooxygenase activity and the interaction of [³H] BaP metabolites with cellular DNA and also modulated BaP induced inhibition of hormone synthesis in GH cells.     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.