Calcium mobilisation modulates growth of lens cells.

The modulating effect of calcium cell signalling agonists on tissue growth was studied in a rabbit lens cell line (NN1003A). Calcium mobilisation was measured after Fura-2 incorporation and growth assayed either by direct Coulter counting or [3H]-thymidine incorporation. Transient increases in cytoplasmic calcium were elicited by rabbit serum, histamine, ATP and PDGF. Thapsigargin induced a prolonged increase and all of the above agonists failed to elicit a response after thapsigargin. Rabbit serum and PDGF both increased cell growth in a concentration-dependent manner. While histamine and ATP had little effect in serum-free medium, they reduced serum-stimulated growth. Acetylcholine and FGF did not produce a marked rise in cytoplasmic calcium and neither did they modulate growth. Both thapsigargin and caffeine greatly inhibited growth. These findings indicate that, in lens cells, agonists that mobilise calcium, whether by acting through G-protein or tyrosine kinase receptors, also modulate lens cell growth. Agents such as thapsigargin and caffeine that inactivate the same calcium store also inhibit growth.

Role of the endoplasmic reticulum in shaping calcium dynamics in human lens cells.

PURPOSE: Localized cortical cataracts in the human lens have been shown to involve a selective increase in calcium with no change in sodium content. Recent studies in the rat lens in vitro have shown that the store-operated channel is highly selective for calcium over sodium, and therefore this channel was characterized further in human lens cells. METHODS: Human primary cultures were initiated from epithelial explants and passaged onto coverslips. After incorporating Fura-2, agonist- or thapsigargin-induced changes in cytosolic calcium were monitored and calibrated using fluorometric digital imaging techniques. RESULTS: Histamine and adenosine triphosphate (ATP; 10 microM) induced a large transient increase in cytosolic calcium followed by a maintained lower plateau phase in the continued presence of the calcium-signaling agonist. The second phase was abolished by removing external calcium and represented the contribution from the store-operated influx. The store-operated pathway was blocked by inorganic agents such as zinc and nickel (100 microM) but was insensitive to the voltage-sensitive calcium channel blocker, nifedipine (1 mM). Depolarizing the membrane voltage by raising the external potassium (75 mM) also blocked the influx. Similar results were obtained if the store was first emptied directly using thapsigargin (1 microM), and with this agent it was also possible to observe the very slow activation and inactivation kinetics (>10 seconds) of the channel. Addition of manganese to the bathing medium initiated a quench of Fura-2 isobestic fluorescence that was enhanced 2.9 +/- 0.3-fold after 10 microM ATP addition. There was a delay of 82 +/- 16 seconds between initiation of the calcium spike and the Mn2+ quench rate, indicating the presence of a delayed entry pathway. In the resting state, removal of, or increasing extracellular calcium concentration 10-fold did not perturb the level of cytosolic Ca2+. Similar maneuvers performed after agonist- or thapsigargin-induced store depletion of intracellular stores brought about dramatic changes in cytosolic Ca2+ consistent with the activation of a Ca2+ entry pathway. Lower concentrations of agonist induced oscillations of Ca2+ that continued for a short time in Ca-free solution. No increase in Mn2+ quench rate was associated with oscillations. A 100-microM zinc- and KCl-induced blockade of Ca2+ entry had no effect on the form of agonist-induced oscillations. Inhibition of Ca2+ influx by zinc (100 microM) converted a sustained Ca2+ response to a train of repetitive Ca2+ spikes. CONCLUSIONS: Human lens cells normally have very low Ca2+ permeability. Depletion of intracellular stores by agonists or thapsigargin initiates a Ca2+ entry pathway that is not required for the Ca2+ oscillations induced by low concentrations of agonist. This potentially provides a signal transduction mechanism with minimal risk of Ca2+ overload to the lens, whereas overactivation of the store-operated channel is a possible way of increasing calcium in the lens and could explain the distribution found in localized cataracts.

Histamine and ATP mobilize calcium by activation of H1 and P2u receptors in human lens epithelial cells.

1. Cytoplasmic calcium concentration ([Ca2+]1) of single superfused tissue-cultured human lens epithelial cells (HLEC) was monitored using the fluorescent dye fura-2; the resting values were low and stable for several hours ([Ca2+]i = 96 +/- 20 nM; mean +/- S.D., n = 16). 2. Continuous superfusion with either ATP or histamine (0.1-10 microM) produced regular oscillations in [Ca2+]i that could be maintained for a short time in the absence of external calcium. 3. Short (30 s) pulses of histamine (0.1-100 microM) induced a transient rise in [Ca2+]i, the time course of which was insensitive to the removal of external calcium. The rate of rise and the amplitude of the response were very sensitive to agonist concentration, whereas the rate of recovery was relatively constant. 4. The responses to long pulses of histamine (> 100 s) consisted of an initial transient followed by a maintained [Ca2+]i which returned to baseline on removal of external calcium. 5. The kinetics of the responses to short and long pulses of ATP (0.1-100 microM) were very similar to those of histamine and showed a similar sensitivity to the presence or absence of external calcium. 6. The histamine responses were abolished by triprolidine (1 microM), but unaffected by ranitidine (1 microM), indicating that an Hi receptor subtype is activated by histamine. 7. The ATP responses were reversibly inhibited by suramin and the potency sequence for a range of agonists was ATP = UTP = ATP gamma S > ADP = GTP >> AMP = adenosine, indicating that activation of a P2u receptor subtype was responsible for the increase in [Ca2+]i. 8. Both histamine and ATP responses were abolished by thapsigargin (100 nM), confirming that calcium release from intracellular stores was responsible for the initial peak of the response. Application of either agonist during the plateau phase of the thapsigargin response often led to a marked, but reversible, decline in [Ca2+]i, indicating the presence of a further, normally hidden, calcium regulatory factor associated with the presence of the agonist. 9. Maximal concentrations of either histamine or ATP totally emptied the calcium store as a subsequent application of the other agonist (or thapsigargin), in the absence of external calcium, failed to induce a further increase in the calcium signal.